Methods of treating nervous system diseases

Abstract

Provided herein are methods of promoting clearance of beta-amyloid (A beta) plaques, methods of reducing neuroinflammation, and methods of treating neurological diseases (e.g., Alzheimer's disease) with certain bispecific antibodies or antigen-binding fragments, one of these bispecific antibodies or antigen binding fragments is specific to internalizing antigens expressed on the surface of nerve cells and the other is specific to toxic forms of A [beta] protein. Exemplary antibodies, features thereof, and methods of screening for other therapeutic bispecific antibodies are also described herein.

Description

[0001] 1. Priority

[0002] This application claims priority to U.S. Provisional Application No. 63 / 333,308, filed on April 21, 2022, the entire contents of which are incorporated herein by reference.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing named "SBL010PCTSL.xml" that complies with WIPO standard ST.26, created on March 29, 2023, with a size of 171,282 bytes, the entire contents of which are incorporated herein by reference. 3. Technical field

[0005] The present invention relates to molecular biology and neurobiology, and in particular to the identification and use of antibodies in the treatment of various neurological diseases associated with amyloid-β and / or neuroinflammation, such as Alzheimer's disease, amyloidosis and β-amyloid pathology. 4. Background Technology

[0006] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by extracellular plaques containing amyloid β (Aβ). The causes of these conditions are generally unknown. It has been shown that the removal of Aβ plaques and possible other forms, including but not limited to soluble oligomers, protofibrils, fibrils (N-terminal truncated forms and post-translationally modified forms) of Aβ that trigger plaque formation can bring clinical benefits to AD patients. However, there are limited therapeutic options that can be used to remove Aβ plaques, which are often associated with vascular side effects such as ARIA-E and ARIA-H. Therefore, new methods for more effectively removing Aβ plaques and AD treatment options with fewer side effects are highly desired. The methods provided in the present disclosure meet this need and provide related advantages. 5. Summary of the invention

[0007] Provided herein are methods for promoting clearance of amyloid-β (Aβ) plaques and possibly other forms thereof, including but not limited to soluble oligomers, protofibrils, protofibrils (N-terminally truncated forms and post-translationally modified forms) (collectively referred to as Aβ) that trigger plaque formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to a surface receptor or antigen expressed on neurons, glial cells or endothelial cells at one end and specifically binds to Aβ at the other end, wherein the surface receptor of the bispecific antibody or antigen-binding fragment / antigen binding portion (a) induces internalization of surface receptors / antigens; and / or (b) inhibits the activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes or other neuroimmune cells; and / or (d) regulates antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells.

[0008] Also provided herein is a method for reducing neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to a surface receptor or antigen expressed on neural cells at one end and specifically binds to Aβ at the other end, wherein the surface receptor / antigen-binding portion of the bispecific antibody or antigen-binding fragment (a) induces internalization of the surface receptor / antigen; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells.

[0009] In some embodiments of the methods provided herein, the subject has clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disease caused by pathogenic Aβ peptides in blood vessels.

[0010] Also provided herein is a method for treating an Aβ-related disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to a surface receptor or protein expressed on neural cells at one end and specifically binds to Aβ at the other end, wherein the surface receptor / antigen-binding portion of the bispecific antibody or antigen-binding fragment targeting neural cells (a) induces internalization of the surface receptor / antigen; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation by microglia, astrocytes, endothelial cells, or other neuroimmune cells to infiltrating T cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells.

[0011] Also provided herein is a method for treating a disease or disorder associated with neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to a surface receptor or antigen expressed on a neural cell at one end and specifically binds to Aβ at the other end, wherein the surface receptor / antigen-binding portion of the bispecific antibody or antigen-binding fragment that targets a neural cell (a) induces internalization of the surface receptor / antigen; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neural immune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neural immune cells; and / or (d) regulates antigen presentation by microglia, astrocytes, endothelial cells, or other neural immune cells to infiltrating T cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neural immune cells.

[0012] In some embodiments, the Aβ-related or neuroinflammation-related disease or disorder is clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disease caused by pathogenic Aβ peptides in blood vessels. In some embodiments, the Aβ-related disease or disorder is Alzheimer's disease.

[0013] In some embodiments, the antibody or antigen-binding fragment used in the methods described herein is a bispecific antibody or antigen-binding fragment.

[0014] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein is a bispecific antibody in the form selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. In some embodiments, the bispecific antibody is in the form of an IgG1 antibody.

[0015] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein comprises a surface receptor / antigen-binding portion of an antibody format selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody, and the Aβ binding antibody is in the form of a single-chain Fv (scFv). In some embodiments, the bispecific antibody is in the form of an IgG1 antibody.

[0016] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein comprises an Aβ binding portion in the form of an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody, while the surface receptor / antigen-binding antibody is in the form of a single-chain Fv (scFv). In some embodiments, the bispecific antibody is in the form of an IgG1 antibody.

[0017] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single domain antibody (sdAb) and heavy chain antibody (HCAb).

[0018] In some embodiments, the bispecific antibodies or antigen-binding bodies used in the methods disclosed herein are chimeric antibodies or antigen-binding bodies, humanized antibodies or antigen-binding bodies, or human antibodies or antigen-binding bodies.

[0019] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to a surface receptor or antigen expressed on neural cells at one end and specifically binds to Aβ protein at the other end.

[0020] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to a surface receptor or antigen expressed on microglia at one end and specifically binds to Aβ protein at the other end.

[0021] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to human CD22 expressed on neural cells at one end and specifically binds to Aβ protein at the other end.

[0022] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds CLLNFSCYGYPIQ (SEQ ID NO: 122) and VFTRSELKFSPQWSHHGKIVTC (SEQ ID NO: 123) expressed on CD22 on neural cells at one end and specifically binds Aβ protein at the other end.

[0023] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to human CD22 expressed on microglia at one end and specifically binds to Aβ protein at the other end.

[0024] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds CLLNFSCYGYPIQ (SEQ ID NO: 122) and VFTRSELKFSPQWSHHGKIVTC (SEQ ID NO: 123) expressed on microglia to human CD22 at one end and specifically binds Aβ protein at the other end.

[0025] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to human CD33 in neural cells at one end and specifically binds to Aβ protein at the other end.

[0026] In some embodiments, the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein specifically bind to human CD33 in microglia at one end and specifically bind to Aβ protein at the other end.

[0027] In some embodiments, the bispecific antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to human CD74 in neural cells at one end and specifically binds to Aβ protein at the other end.

[0028] In some embodiments, the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein specifically bind to human CD74 in microglia at one end and specifically bind to Aβ protein at the other end.

[0029] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0030] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a chimeric antibody or antigen binding fragment. In some embodiments, the VL and VH of the chimeric antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the chimeric antibody or antigen binding fragment is SM03.

[0031] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM06.

[0032] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0033] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a chimeric antibody or antigen binding fragment. In some embodiments, the VL and VH of the chimeric antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In some embodiments, the chimeric antibody or antigen binding fragment is LL2.

[0034] In some embodiments, the surface receptor / antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment specific for human CD22. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively. In some embodiments, the humanized antibody or antigen binding fragment is hLL2.

[0035] In some embodiments, the receptor binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

[0036] In some embodiments, the surface receptor or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Gemtuzumab.

[0037] In some embodiments, the receptor binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34, respectively.

[0038] In some embodiments, the surface receptor or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively. In some embodiments, the humanized antibody or antigen binding fragment is HuMy9-6.

[0039] In some embodiments, the receptor binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively.

[0040] In some embodiments, the surface receptor or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 44, respectively. In some embodiments, the humanized antibody or antigen binding fragment is lintuzumab.

[0041] In some embodiments, the receptor binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively.

[0042] In some embodiments, the surface receptor or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a chimeric antibody or antigen binding fragment. In some embodiments, the VL and VH of the chimeric antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 52, respectively. In some embodiments, the chimeric antibody or antigen binding fragment is LL1.

[0043] In some embodiments, the surface receptor or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 53 and SEQ ID NO: 54, respectively. In some embodiments, the humanized antibody or antigen binding fragment is hLL1.

[0044] In some embodiments, the amyloid β binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, respectively.

[0045] In some embodiments, the amyloid beta or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 61 and SEQ ID NO: 62 or SEQ ID NO: 63, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Aducanumab.

[0046] In some embodiments, the amyloid β binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69, respectively.

[0047] In some embodiments, the amyloid beta or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 71, respectively. In some embodiments, the humanized antibody or antigen binding fragment is BAN2401.

[0048] In some embodiments, the amyloid β portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, respectively.

[0049] In some embodiments, the amyloid beta or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 78 and SEQ ID NO: 79, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Gantenerumab.

[0050] In some embodiments, the amyloid β binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85, respectively.

[0051] In some embodiments, the amyloid beta or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 86 and SEQ ID NO: 87, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Crenezumab.

[0052] In some embodiments, the amyloid β binding portion of the bispecific antibodies or antigen-binding fragments used in the methods disclosed herein comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93, respectively.

[0053] In some embodiments, the amyloid beta or antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the VL and VH of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 94 and SEQ ID NO: 95, respectively. In some embodiments, the humanized antibody or antigen binding fragment is AD38.

[0054] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 96 and SEQ ID NO: 97, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM03-cre.

[0055] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 96 and SEQ ID NO: 98, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM03-ban.

[0056] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 96 and SEQ ID NO: 99, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM03-gan.

[0057] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 96 and SEQ ID NO: 100, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM03-adu.

[0058] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 102, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM06-ban.

[0059] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 103, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM06-gan.

[0060] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 104, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM06-adu.

[0061] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 101 and SEQ ID NO: 105, respectively. In some embodiments, the humanized antibody or antigen binding fragment is SM06-ad38.

[0062] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 106 and SEQ ID NO: 107, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Gem-adu.

[0063] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 108 and SEQ ID NO: 109, respectively. In some embodiments, the humanized antibody or antigen binding fragment is 96-adu.

[0064] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 110 and SEQ ID NO: 111, respectively. In some embodiments, the humanized antibody or antigen binding fragment is Lin-adu.

[0065] In some embodiments, the antigen binding portion of the bispecific antibody or antigen binding fragment used in the methods disclosed herein is a humanized antibody or antigen binding fragment. In some embodiments, the light chain and heavy chain of the humanized antibody or antigen binding fragment have the amino acid sequences of SEQ ID NO: 112 and SEQ ID NO: 113, respectively. In some embodiments, the humanized antibody or antigen binding fragment is hLL1-adu.

[0066] In some embodiments of the methods disclosed herein, the bispecific antibody or antigen-binding fragment is administered intravenously, intramuscularly, subcutaneously, intracranial, intrathecally, intraventricularly, intraperitoneally, intranasally, parenterally, topically, or intradermally. In some embodiments, the bispecific antibody or antigen-binding fragment is administered intravenously. In some embodiments, the bispecific antibody or antigen-binding fragment is administered subcutaneously.

[0067] In some embodiments of the methods disclosed herein, the bispecific antibody or antigen binding fragment is administered in a therapeutically effective amount within the range of 1-50 mg / kg subject weight. In some embodiments, the therapeutically effective amount is about 1, about 2, about 3, about 5, about 10, about 15 or about 30 mg / kg subject weight. In some embodiments, the bispecific antibody or antigen binding fragment is administered in a therapeutically effective amount of 300-1200 mg per dose. In some embodiments, the bispecific antibody or antigen binding fragment is administered once every two weeks or monthly. In some embodiments, the bispecific antibody or antigen binding fragment is administered in multiple doses. In some embodiments, the bispecific antibody or antigen binding fragment is administered in multiple doses over a period of at least three months, at least six months or at least one year.

[0068] In some embodiments of the methods disclosed herein, the bispecific antibody or antigen binding fragment is administered in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-β-amyloid antibody, an anti-CD22 antibody, an anti-CD33 antibody, an anti-CD74 antibody, a Tau aggregation inhibitor, a Tau protein modulator, a cholinesterase inhibitor, an acetylcholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, a β-secretase inhibitor, or an insulin sensitizer.

[0069] In some embodiments, the second therapeutic agent is an anti-β-amyloid antibody selected from the group consisting of aducanumab, donanemab, gantenerumab, lecanemab, crenezumab, bapineuzumab, solanezumab, and AD-38.

[0070] In some embodiments, the second therapeutic agent is an anti-CD22 antibody selected from the group consisting of SM03, SM06, Epratuzumab, Moxetumomab pasudotox, inotuzumab ozogamicin, OXS-1550, SCRI-CAR22v2, MB-CAR-T19-22, CD22-CART, AUTO-3, UCART-22, TRPH-222, JCAR-018, ThisCART22, JNJ-75348780, YT-19 / 22, AUTO-1-NG, CTA-101, GC-022, LB-1909, MendCART, JJO-686, SCRI-CAR19X22v1, Hb22.7, and anti-CD22-NMS-249.

[0071] In some embodiments, the second therapeutic agent is an anti-CD33 antibody selected from the group consisting of AL003, gemtuzumab ozogamicin, lintuzumab, vadastuximab talirine, BI836858, VCAR-33, VOR-33, OXS-3550, AMV-564, eluvixtamab, LB-1910, JNJ-67571244, PRGN-3006, ICG-136, and GEM-333.

[0072] In some embodiments, the second therapeutic agent is an anti-CD74 antibody selected from the group consisting of milatuzumab, STRO-001, hLL1-CL2A-SN-38, milatuzumab-Fab-veltuzumab, 74-(20)-(20), 2L-rpRNAse-milatuzumab-g4P, milatuzumab-doxorubicin conjugate, and HuMax-CD74-ADC.

[0073] In some embodiments of the methods disclosed herein, the subject is a human subject. 6. Description of the drawings

[0074] Figure 1A and Figure 1B Flow cytometry binding data are provided, which show that CD22 ( Figure 1A ) and CD33 and CD74 ( Figure 1B ) in the human microglial cell line HMC-3.

[0075] Figure 2 Octet in vitro binding data are provided, which demonstrates the binding of Aβ1-42 to recombinant CD22 protein. As shown, the K of CD22 binding to oligomeric Aβ1-42 is D It is 2.79nM.

[0076] Figure 3 SDS-PAGE data on the size of CD22-Aβ bispecific antibodies SM03-Adu and SM06-Adu are presented.

[0077] Figure 4 ELISA data are provided demonstrating binding of bispecific antibodies SM03-Adu, SM06-Adu, SM03-BAN, SM06-BAN to CD22 and Aβ.

[0078] Figure 5 Flow cytometry data are provided, demonstrating binding of the CD22-Aβ bispecific antibody SM03-Adu to HMC-3 cells.

[0079] Figure 6 Depicted is the enhanced inhibition of TNF-α expression by SM03-BAN compared to anti-Aβ antibody (BAN 2401) or anti-CD22 antibody (SM03) alone.

[0080] Figure 7 It was shown that SM03-Adu had enhanced inhibitory effect on IL-6 release compared with anti-Aβ antibody (aducanumab) and anti-CD22 antibody (SM03).

[0081] Figure 8 Shown are the inhibitory effects of bispecific antibodies SM03-Adu and SM06-Adu on Aβ-induced IL-1β release in human PBMCs.

[0082] Fig. 9 The bispecific antibodies SM03-Adu and SM06-Adu are shown to inhibit Aβ-induced oligodendrocyte death.

[0083] Fig.10 The internalization rates of the bispecific antibodies SM03-Adu, SM06-Adu, hLL1-Adu and Gem-Adu are shown.

[0084] Fig.11Shown is a comparison of Aβ clearance of two different versions of SM03-Adu with anti-Aβ monoclonal antibody (aducanumab) or SM03 alone. 7. Specific implementation methods

[0085] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by extracellular plaques containing amyloid beta (Aβ). There is still an urgent need for effective treatment options. Disclosed herein is the surprising discovery of the inventors that bispecific antibodies targeting surface receptors named CD22, CD33 or CD74 and oligomeric or fibrillar Aβ1-42 promote the clearance of Aβ plaques by inducing surface receptor internalization. In addition, it was found that the bispecific antibodies disclosed herein can also inhibit autoimmunity in the central nervous system (CNS), thereby providing further therapeutic benefits. Therefore, in some embodiments, provided herein are methods for reducing Aβ accumulation and neuroinflammation through a CD22 / CD33 / CD74-mediated mechanism, as well as treating related diseases or disorders (such as AD).

[0086] It is estimated that by 2050, the number of people with dementia worldwide will increase from 50 million in 2010 to 113 million. AD is the leading cause of dementia and is often complicated by other neurodegenerative and cerebrospinal pathologies. Neuropathological features begin 15 to 20 years before the onset of overt cognitive symptoms. AD patients will progress from normal baseline cognitive abilities to overt brain dysfunction symptoms through subtle changes in the preclinical stage, known as prodromal AD, and finally to AD dementia, which ultimately impairs their previously independent ability to perform activities of daily living (ADL) due to cognitive deficits. The preclinical stage of AD is generally considered to be the stage that occurs before a clinical diagnosis of cognitive impairment is considered. AD dementia is a synaptic dysfunction disease that includes changes at the molecular, cellular, and connectome levels. The clinical manifestations of AD patients include dementia with amnesia or non-amnesia symptoms. It is often accompanied by impairments in speech, visual-spatial processing, and executive function. (Scharre, Practical Neurology (2019); Dubois et al., Alzheimers Dement (2016), 12 (3): 292-323.) The neuropathological hallmarks of AD are characterized by excessive neuroinflammation, extracellular Aβ accumulation containing plaques, and hyperphosphorylated tau protein. Aβ plaques are widely present in the cortex and hippocampus, and lead to the destruction of neuronal networks by triggering neuronal loss and promoting neuroinflammation through microglia.

[0087] Aβ is derived from amyloid precursor protein, a transmembrane protein that is abundant in the neuronal membrane. Physiologically, it is cleaved by α-secretase and then by γ-secretase to form APPsα. It regulates synaptic strength by regulating calcium flux and potassium channels. In the amyloidogenic pathway, Aβ is cleaved by β-secretase and then by γ-secretase to form Aβ1-42. Aβ1-42 misfolds into a β-sheet conformation and aggregates into toxic oligomeric forms. According to the amyloid cascade hypothesis, toxic oligomeric Aβ1-42 aggregates into plaques that lead to neurotoxicity and dementia. Oligomeric Aβ1-42 directly binds to metabotropic glutamate receptor 5 (mGluR5), N-methyl-D-aspartate (NMDA) receptors, and other neuronal receptors such as α-7 nicotinic acetylcholine receptors and insulin receptors to induce pathological changes in synaptic strength and dendritic spine morphology. Oligomeric Aβ1-42 can also stimulate microglia through pattern recognition receptors, namely Toll-like receptors (TLR), Nod-like receptors (NLR), RIG-like receptors (RLR), AIM2-like receptors (ALR), to trigger neuroinflammation. Aβ1-42 induces microglia to release proinflammatory cytokines, namely IL-1β, IL-8, and TNFα. In addition, it activates the NLRP3 inflammasome cascade to secrete ASC speck protein (apoptosis-associated speck-like protein containing caspase-1 recruitment domain), which further aggregates Aβ1-42 into nuclei.

[0088] It has been proposed that clearance of toxic oligomeric Aβ could lead to clinical benefit in AD patients. As in 2021, of the 17 disease-modifying therapies in Phase III, 29% have Aβ as their mechanism of action (MOA). Anti-Aβ antibodies, including aducanumab, doranizumab, gantnerumab, lecanimumab, cranizumab, bapineumab, and sorazumab, target various toxic forms of Aβ through Fc receptor-mediated phagocytosis and non-Fc receptor-mediated Aβ clearance.

[0089] Aducanumab is an IgG1 mAb that selectively targets soluble oligomers and insoluble fibrils of Aβ. Aducanumab binds to amino acids 3-6 and recognizes a conformational epitope on aggregated Aβ, but not the monomeric form. Binding of the antibody to Aβ aggregates triggers clearance via Fcγ receptor-mediated phagocytosis, thereby restoring calcium balance in neuronal networks in AD patients. Aducanumab was approved by the FDA in June 2021 for the treatment of AD as clinical studies have consistently shown a reduction in amyloid plaques.

[0090] In addition to AD, other diseases or disorders associated with abnormal accumulation of Aβ include, for example, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular diseases caused by pathogenic Aβ peptides in blood vessels.

[0091] Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the particular embodiments described herein and that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

[0092] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. In general, the terms used in connection with cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization described herein and these techniques are all commonly used terms and techniques well known in the art.

[0093] The term "a" or "an" entity refers to one or more entities; for example, "a vector" is understood to mean one or more vectors.

[0094] The term "and / or" as used herein should be understood to specifically disclose each of the two specified features or components, whether or not the other is included. Therefore, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0095] Range: Throughout this disclosure, various aspects of the invention may be expressed in range format. It should be understood that description in range format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of the invention. Therefore, description of a range should be considered to have specifically disclosed all possible subranges within that range as well as individual values. For example, description of a range of 1 to 6 should be considered to have specifically disclosed subranges of 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0096] Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and / or SEQ ID NOS. It should be understood that one skilled in the art can readily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0097] 7.1 Clearing Aβ and Reducing Neuroinflammation

[0098] A variety of receptor proteins have been found on the surface of neural cells such as microglia, astrocytes, and oligodendrocytes, which play a role in regulating the immune system of the CNS. Of particular interest is that some of these receptors, such as human CD22, CD33, and CD74, can also induce receptor internalization. Bispecific antibodies targeting these receptors at one end and Aβ protein at the other end will have the dual functions of regulating pro-inflammatory activity and inducing Aβ internalization and clearance, thereby enhancing the efficacy of treating neurological diseases.

[0099] CD22 is a B cell restricted antigen, which belongs to the immunoglobulin (Ig) superfamily.CD22 is a type I transmembrane sialic acid glycoprotein, also known as polysialic acid binding immunoglobulin type lectin (Siglecs).Human CD22 specifically binds to the structure N-acetylneuraminic acid α (2-6) galactose (NeuAc-α (2-6) Gal), which is known to be expressed on hematopoietic cells, hepatocytes, lung epithelial cells, spleen cords and splenic nodules, ileostomy, cardiac interstitium, blood vessels, skin epithelial cells secreted on eccrine sweat glands, colon stromal cells, liver sinusoids and stromal cells, and cells in the central nervous system (CNS) such as microglia in the white matter of the brain (Gagneux et al., Journal of Biological Chemistry (2003), 278 (48): 48245-50; Safaiyan et al., Neuron (2021), 109 (7): 1100-17.e10). CD22 binds to specific ligands distributed on the same cell (cis binding) or on different cells (trans binding). Cis binding of CD22 is often seen between adjacent molecules, where the glycan binding site of a CD22 molecule is linked to the glycan molecule of another CD22 or glycoprotein on the same cell, forming homo-oligomers or homo-multimers. Cis binding of CD22 has been shown to have a masking effect on the molecule, preventing Siglec from forming cell-to-cell connections (trans binding). In resting B cells, CD22 is an important cis ligand of itself, which can form CD22 homo-oligomers. Trans-binding of CD22 results in physical association of Siglec with the B cell receptor (BCR), resulting in a maximal inhibitory response that is required for achieving immune tolerance by stimulating inhibitory immune signals (Pessutto et al., 1987. J. Immunol. 138:98-103; Doody et al., 1995. Science 269:242-344; Lanoue et al., 2002. Eur. J. Immunol. 32:348-355; Courtney et al., 2009. PNAS 106:2500-2505). Additional information about human CD22, including its exemplary amino acid sequence, can be found in public databases such as UniProt (UniProt KB / Swiss-Prot ID: P20273) and GENBANK (NCBI Ref. NP_001172028.1, NP_001172029.1, NP_001172030.1, NP_001265346.1, NP_001762.2). Exemplary sequences are also provided below.

[0100] 1MHLLGPWLLL LVLEYLAFSD SSKWVFEHPE TLYAWEGACV WIPCTYRALD GDLESFILFH

[0101] 61NPEYNKNTSK FDGTRLYEST KDGKVPSEQK RVQFLGDKNK NCTLSIHPVH LNDSGQLGLR121MESKTEKWME RIHLNVSERP FPPHIQLPPE IQESQEVTLT CLLNFSCYGY PIQLQWLLEG

[0102] 181VPMRQAAVTS TSLTIKSVFT RSELKFSPQW SHHGKIVTCQ LQDADGKFLS NDTVQLNVKH

[0103] 241TPKLEIKVTP SDAIVREGDS VTMTCEVSSS NPEYTTVSWL KDGTSLKKQN TFTLNLREVT

[0104] 301KDQSGKYCCQ VSNDVGPGRS EEVFLQVQYA PEPSTVQILH SPAVEGSQVE FLCMSLANPL

[0105] 361PTNYTWYHNG KEMQGRTEEK VHIPKILPWH AGTYSCVAEN ILGTGQRGPG AELDVQYPPK421KVTTVIQNPM PIREGDTVTL SCNYNSSNPS VTRYEWKPHG AWEEPSLGVL KIQNVGWDNT481TIACAACNSW CSWASPVALN VQYAPRDVRV RKIKPLSEIH SGNSVSLQCD FSSSHPKEVQ

[0106] 541FFWEKNGRLL GKESQLNFDS ISPEDAGSYS CWVNNSIGQT ASKAWTLEVL YAPRRLRVSM601SPGDQVMEGK SATLTCESDA NPPVSHYTWF DWNNQSLPYH SQKLRLEPVK VQHSGAYWCQ661GTNSVGKGRS PLSTLTVYYS PETIGRRVAV GLGSCLAILI LAICGLKLQR RWKRTQSQQG

[0107] 721LQENSSGQSF FVRNKKVRRA PLSEGPHSLG CYNPMMEDGI SYTTLRFPEM NIPRTGDAES

[0108] 781SEMQRRPPPDC DDTVTYSALH KRQVGDYENV IPDFPEDEGI HYSELIQFGV GERPQAQENV

[0109] 841DYVILKH (SEQ ID NO: 114)

[0110] CD33 is a type I transmembrane sialoglycoprotein, also known as polysialic acid-binding immunoglobulin-type lectins (Siglecs). Human CD33 is expressed on hematopoietic and phagocytic cells, i.e., hematopoietic progenitor cells, granulocyte and monocytic precursors, macrophages, monocytes, dendritic cells, and microglia. Human CD33 preferentially binds to N-acetylneuraminic acid α(2-6) galactose (NeuAc-α(2-6)Gal). Upon ligand binding, the ITIM motif of human CD33 is phosphorylated and recruits SHP phosphatases. Subsequently, activation signaling pathways initiated by ITAM-containing receptors are downregulated. CD33 has been identified as an AD-associated gene: SNPrs3865444C is an AD risk allele that promotes CD33 expression in microglia and limits Aβ phagocytosis; while SNP rs3865444A is an AD protective allele that results in the production of truncated CD33 lacking the sialic acid binding domain (D2). The resulting truncated CD33 is not expressed on the membrane surface and resides in the cytoplasm. Additional information about human CD33, including its exemplary amino acid sequence, can be found in public databases such as UniProt (UniProt KB / Swiss-Prot ID: P20138) and GENBANK (NCBI Ref. NP_001076087.1, NP_001171079.1, NP_001763.3). Exemplary sequences are also provided below.

[0111] 1MPLLLLLPLL WAGALAMDPN FWLQVQESVT VQEGLCVLVP CTFFHPIPYY DKNSPVHGYW61FREGAIISRD SPVATNKLDQ EVQEETQGRF RLLGDPSRNN CSLSIVDARR RDNGSYFFRM

[0112] 121ERGSTKYSYK SPQLSVHVTD LTHRPKILIP GTLEPGHSKN LTCSVSWACE QGTPPIFSWL

[0113] 181SAAPTSLGPRTTHSSVLIIT PRPQDHGTNL TCQVKFAGAG VTTERTIQLN VTYVPQNPTT

[0114] 241GIFPGDGSGK QETRAGVVHG AIGGAGVTAL LALCLCLIFF IVKTHRRKAA RTAVGRNDTH

[0115] 301PTTGSASPKH QKKSKLHGPT ETSSCSGAAP TVEMDEELHY ASLNFHGMNP SKDTSTEYSE

[0116] 361VRTQ(Seq No.115)

[0117] Human CD74 is a type II single-span transmembrane protein with 4 isoforms. CD74 is found to be expressed in lymphoid and myeloid cell lineages such as B cells, T cells, monocytes, macrophages, and dendritic cells. CD74 acts as a chaperone for MHCII proteins and regulates antigen presentation, endosomal trafficking, dendritic cell migration, and macrophage migration inhibitory factor (MIF) signaling cascades. CD74 is also expressed in human microglia and is upregulated in neurofibrillary tangles in Alzheimer's disease. CD74 has been shown to interact with amyloid precursor protein and inhibit Aβ production. Additional information about human CD74, including exemplary amino acid sequences thereof, can be found in public databases such as UniProt (UniProt KB / Swiss-Prot ID: P04233) and GENBANK (NCBI Ref. NP_001020329.1, NP_001020330.1, NP_001351012.1, NP_001351013.1, NP_004346.1). Exemplary sequences are also provided below.

[0118] 1MHRRRSRSCR EDQKPVMDDQ RDLISNNEQL PMLGRRPGAP ESKCSRGALY TGFSILVTLL

[0119] 61LAGQATTAYF LYQQQGRLDK LTVTSQNLQL ENLRMKLPKP PKPVSKMRMA TPLLMQALPM

[0120] 121GALPQGPMQN ATKYGNMTED HVMHLLQNAD PLKVYPPLKG SFPENLRHLK NTMETIDWKV

[0121] 181FESWMMHWLL FEMSRHSLEQ KPTDAPPKES LELEDPSSGL GVTKQDLGPV PM(SeqNo.116)

[0122] CD22 is moderately expressed on microglia, which are the main antigen-presenting cells in the CNS. The presented antigens (e.g., Aβ or other self-antigens) can be recognized by infiltrating regulatory T cells, which inhibit the activation of microglia by secreting IL-10. The inventors previously disclosed that certain anti-CD22 antibodies can induce CD22 internalization in human microglia. It was also found that CD22 binds to oligomeric Aβ1-42, and the internalization of CD22 can promote the clearance of Aβ by phagocytosis of CD22-bound Aβ1-42 and macrophagocytosis of soluble Aβ.

[0123] Specific antibodies for CD22, CD33, and CD74 have been found to induce rapid internalization of antigen-binding antibodies. In the present disclosure, the inventors take advantage of the presence of these internalized antigens on microglia, astrocytes, and oligodendrocytes, and propose an invention using bispecific antibodies that specifically bind to CD22, CD33, or CD74 at one end and Aβ at the other end to enhance the effect of eliminating amyloid plaques. Therefore, in some embodiments, provided herein is a method for promoting the removal of Aβ using bispecific antibodies targeting CD22, CD33, or CD74 and Aβ disclosed herein. In some embodiments, provided herein is a method for treating Aβ-related diseases or disorders using bispecific antibodies disclosed herein. In some embodiments, the Aβ-related disease or disorder is AD.

[0124] As used herein and understood in the art, "internalization" of CD22, CD33 or CD74 refers to the endocytosis of CD22, CD33 or CD74, i.e., the process of invagination and engulfment of CD22, CD33 or CD74 molecules by cells. There are three types of endocytosis processes: pinocytosis, phagocytosis and receptor-mediated endocytosis. Pinocytosis refers to the process by which a cell engulfs a small amount of extracellular fluid (with possible small particles) into the cell through the process of invagination, also known as "cell-drinking". Phagocytosis refers to the process by which a cell engulfs relatively large molecules or organisms (such as bacterial cells). Receptor-mediated endocytosis refers to the process by which a substance directly binds to a receptor protein on the cell membrane, thereby initiating the invagination process and forming a layer of protein covering layer called a clathrin coating. The vesicles formed during receptor-mediated endocytosis contain this clathrin protein coating. CD22, CD33 or CD74 on the cell membrane is internalized after binding to a specific antibody via clathrin-mediated endocytosis.

[0125] Current anti-Aβ antibody therapies for the treatment of AD rely primarily on FcγR-mediated phagocytosis to reduce the burden of amyloid, but these therapies are associated with undesirable side effects. Specifically, magnetic resonance imaging (MRI) abnormalities, such as signal changes representing "vasogenic edema" (VE) and microhemorrhages (mH), were observed in patients administered amyloid-mitigating therapeutics (anti-Aβ antibodies) in a dose-dependent manner. Amyloid-associated imaging abnormalities (ARIA) were proposed by the Alzheimer's Association Research Roundtable Working Group to describe vasogenic edema (ARIA-E) and microhemorrhages and hemosiderosis (ARIA-H). ARIA-E is primarily caused by inflammation and is closely associated with mAbs that bind to the fibrillar form of Aβ. FcγR-mediated inflammation in microglia can play an important role. For example, inflammation mediated by the release of proinflammatory cytokines induced by FcγRs can exacerbate damage to vascular integrity. Cranelizumab, a humanized IgG4 antibody targeting all forms of Aβ, is associated with fewer ARIA-E events due to reduced association of the IgG4 Fc region with FcγRs on microglia compared with, for example, IgG1 Fc.

[0126] Therefore, the bispecific antibodies provided herein that target CD22, CD33 or CD47 at one end and Aβ at the other end provide a new Aβ clearance mechanism that is not only highly efficient, but also related to reducing vascular side effects like ARIA-E and / or ARIA-H. Unlike Aβ targeting antibodies, the bispecific antibodies disclosed herein promote Aβ clearance through CD22, CD33 or CD74 internalization, which does not involve cross-linking of therapeutic antibodies with FcγR, thereby avoiding subsequent proinflammatory responses. Therefore, a method for effectively removing Aβ with reduced vascular side effects is provided herein. In some embodiments, the methods provided herein do not cause vascular side effects.

[0127] The bispecific antibodies targeting CD22 and Aβ disclosed herein (e.g., SM03 or SM06) promote the clearance of Aβ by CD22 internalization, and (a) promote the cis-trans conversion of CD22 and / or (b) induce the internalization of CD22. The bispecific antibodies targeting CD22 and Aβ disclosed herein promote the cis-trans conversion of CD22 by destroying the cis binding of CD22 on the surface of B cells, microglia or oligodendrocytes and accelerating the internalization of surface CD22. When CD22 molecules are internalized with antibodies that destroy cis binding of the same cluster, CD22 molecules are recovered to the cell surface along with the antibodies, where the antibodies sterically hinder further cis binding, thereby promoting the trans binding of the recovered CD22. Therefore, the bispecific antibodies targeting CD22 and Aβ disclosed herein can also promote the cis-trans conversion of 2,6-sialic acid binding of self-tissues by destroying the cis binding of CD22 on B cells, B cell-derived cell lines, and microglia, thereby restoring the immune tolerance of self-tissues and inhibiting the release of proinflammatory cytokines, thereby promoting the immune tolerance of the central and peripheral immune systems. By promoting the trans binding of CD22 to neuronal 2,6-sialic acid, the anti-CD22 antibodies disclosed herein can inhibit synaptic phagocytosis and neuronal death, as well as inhibit NF-κB signaling and interleukin-6 (IL-6) secretion in microglia. Therefore, the methods disclosed herein have the additional therapeutic benefit of reducing neuroinflammation. Therefore, in some embodiments, methods for reducing neuroinflammation using the anti-CD22 antibodies disclosed herein are provided herein. In some embodiments, methods for treating neuroinflammation-related diseases or disorders using the anti-CD22 antibodies disclosed herein are provided herein.

[0128] The bispecific antibodies targeting CD33 and Aβ disclosed herein (eg, Mylot-Aβ, 96-Aβ, or Lin-Aβ) can induce CD33 internalization, and induce SHP-1 phosphorylation and subsequent inhibition of microglial activation.

[0129] The bispecific antibodies targeting CD74 and Aβ disclosed herein (e.g., LL1-Aβ) can induce CD74 internalization and block the MIF signaling cascade. This inhibitory effect on MIF can reduce neuroinflammation of microglia. The anti-CD74 antibodies disclosed herein increase the interaction between CD74 and APP and reduce the production of Aβ.

[0130] Provided herein is a method for promoting clearance of Aβ plaques in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof, one end of which specifically binds to CD22, CD33 or CD74 and the other end of which specifically binds to Aβ, wherein the bispecific antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits the activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes or other neuroimmune cells; and / or (d) regulates antigen presentation of infiltrating T cells by microglia, astrocytes, endothelial cells or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells. Also provided herein is a method for treating an Aβ-related disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that can specifically bind to CD22, CD33, or CD74, wherein the antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation of infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin sheaths by oligodendrocytes or other neuroimmune cells. In some embodiments, the subject is human. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human CD22, CD33, or CD74. In some embodiments, the A[beta]-related disease or disorder is AD. In some embodiments, the A[beta]-related disease or disorder is preclinical AD.

[0131] In some embodiments, provided herein is the use of a bispecific antibody or an antigen-binding fragment thereof for clearing Aβ plaques, one end of which specifically binds to CD22, CD33 or CD47, and the other end specifically binds to Aβ. In some embodiments, provided herein is the use of a bispecific antibody or an antigen-binding fragment thereof for preparing a drug for clearing Aβ plaques, one end of which specifically binds to CD22, CD33 or CD74, and the other end specifically binds to Aβ. In some embodiments, provided herein is the use of a bispecific antibody or an antigen-binding fragment thereof for treating Aβ-related diseases or disorders, one end of which specifically binds to CD22, CD33 or CD74, and the other end specifically binds to Aβ. In some embodiments, provided herein is the use of a bispecific antibody or an antigen-binding fragment thereof for preparing a drug for treating Aβ-related diseases or disorders, one end of which specifically binds to CD22, CD33 or CD74, and the other end specifically binds to Aβ. In the above-mentioned uses, the bispecific antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits the activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation of infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin sheaths by oligodendrocytes or other neuroimmune cells. In some embodiments, Aβ-related diseases or disorders are AD. In some embodiments, Aβ-related diseases or disorders are preclinical AD. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human CD22, CD33, or CD74.

[0132] Under the treatment of CD22, CD33 or CD74 bispecific antibodies disclosed herein, the internalization rate of Aβ can reach 5.86x10 -6 pg / s / cell, as determined by ELISA of Aβ-treated microglial lysates (data not shown). In some embodiments of the uses or methods provided herein, the clearance rate of Aβ is 0.86 pg / s.

[0133] The methods provided herein can treat diseases or disorders associated with Aβ. Aβ-related diseases or disorders can be clinical or preclinical AD, prodromal AD, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular diseases caused by pathogenic Aβ peptides in blood vessels.

[0134] In some embodiments, the methods provided herein can be used to treat AD. As described above and well known in the art, it is known that clearing oligomeric Aβ brings clinical benefits to AD. AD can be clinical AD, preclinical AD or prodromal AD. In some embodiments, the methods provided herein can be used to treat clinical AD. In some embodiments, the methods provided herein can be used to treat preclinical AD. In some embodiments, the methods provided herein can be used to treat prodromal AD.

[0135] In some embodiments, the methods provided herein can be used to treat Down syndrome. In some embodiments, the methods provided herein can be used to treat clinical or preclinical CAA. In some embodiments, the methods provided herein can be used to treat Parkinson's disease. In some embodiments, the methods provided herein can be used to treat multi-infarct dementia. In some embodiments, the methods provided herein can be used to treat cerebral amyloid angiopathy. In some embodiments, the methods provided herein can be used to treat glaucoma. In some embodiments, the methods provided herein can be used to treat preeclampsia. In some embodiments, the methods provided herein can be used to treat cognitive impairment. In some embodiments, the methods provided herein can be used to treat memory loss. In some embodiments, the methods provided herein can be used to treat vascular diseases caused by pathogenic Aβ peptides in blood vessels.

[0136] Also provided herein is a method for reducing neuroinflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof that specifically binds to CD22, CD33 or CD74 in addition to binding to an Aβ protein, wherein the bispecific antibody or antigen-binding fragment (a) induces internalization of a surface receptor / antigen; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells. Also provided herein is a method for treating a disease or disorder associated with neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof, wherein the receptor binding portion specifically binds to CD22, CD33 or CD74, wherein the bispecific antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation of infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin sheaths by oligodendrocytes or other neuroimmune cells. In some embodiments, the methods provided herein prevent synaptic phagocytosis and neuronal death. In some embodiments, the methods provided herein prevent synaptic engulfment and neuronal death by at least 20%, at least 30%, at least 40%, at least more than 50%, or at least more than 60%. In some embodiments, the methods provided herein prevent synaptic engulfment and neuronal death by at least 50%. In some embodiments, the subject is a human. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD22, CD33, or CD74.

[0137] In some embodiments, provided herein are bispecific antibodies or antigen binding fragments thereof that specifically bind to CD22, CD33, or CD74 for use in reducing neuroinflammation. In some embodiments, provided herein are bispecific antibodies or antigen binding fragments thereof that specifically bind to CD22, CD33, or CD74 for use in the preparation of a medicament for reducing neuroinflammation. In some embodiments, provided herein are bispecific antibodies or antigen binding fragments thereof that specifically bind to CD22, CD33, or CD74 for use in the treatment of diseases or disorders associated with neuroinflammation. In some embodiments, provided herein are bispecific antibodies or antigen binding fragments thereof that specifically bind to CD22, CD33, or CD74 for use in the preparation of a medicament for the treatment of diseases or disorders associated with neuroinflammation. In the above-mentioned uses, the bispecific antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits the activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation of infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promotes remyelination of damaged myelin sheaths by oligodendrocytes or other neuroimmune cells. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human CD22, CD33, or CD74.

[0138] In some embodiments, the anti-CD22, CD33 or CD74 bispecific antibodies or antigen binding fragments used in the methods described herein can induce internalization of CD22, CD33 or CD74. In some embodiments, the anti-CD22 bispecific antibodies or antigen binding fragments used in the methods described herein, and the anti-CD33 bispecific antibodies or antigen binding fragments that may be used, can promote the cis-trans conversion of CD22 and CD33. In some embodiments, the anti-CD22 and anti-CD33 bispecific antibodies or antigen binding fragments used in the methods described herein promote the cis-trans conversion of CD22 and CD33 and induce the internalization of CD22 and CD33.

[0139] As used herein, the term "treat" and its grammatical equivalents in relation to a disease or disorder or a subject suffering from a disease or disorder refers to an action of inhibiting, eliminating, alleviating and / or ameliorating the symptoms, symptom severity and / or symptom frequency associated with the disease or disorder being treated. For example, when used in reference to AD, the term "treat" and its grammatical equivalents refers to an action of reducing the severity of the disease, or delaying or slowing the progression of the disease, including but not limited to (a) reducing the number of Aβ plaques, or slowing the rate of accumulation of pathological Aβ; or (b) delaying, ameliorating or minimizing one or more symptoms associated with AD, such as dementia or cognitive impairment.

[0140] As used herein, the term "administer" and its grammatical equivalents refer to the act of delivering or causing a therapeutic agent or pharmaceutical composition to be delivered into a subject by methods described herein or known in the art. The therapeutic agent can be a compound, a polypeptide, or a cell. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered into a subject. Exemplary administration forms include oral dosage forms such as tablets, capsules, syrups, suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); subcutaneous injection (SC), transdermal dosage forms including creams, gels, powders, or patches; oral dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0141] As used herein, the terms "effective amount", "therapeutically effective amount" and their grammatical equivalents refer to the administration of a preparation to a subject, which can be administered alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, in an amount that can produce any detectable, positive effect on any symptom, aspect or feature of a disease, disorder or condition when administered to a subject. The therapeutically effective amount can be determined by measuring the relevant physiological effects. The exact dosage varies from person to person, depending on the age, weight and general condition of the subject, the severity of the disease being treated, the judgment of the clinician, etc. The therapeutically effective amount is also the amount in which the toxic or harmful effects of the therapeutic agent outweigh the beneficial effects of the treatment. In any case, the appropriate "effective amount" may vary depending on factors such as disease state, age, sex and weight, and can be determined by ordinary technicians in the field through routine experiments. "Prophylactically effective amount" refers to the effective amount required to achieve the desired preventive effect, for example, to delay or prevent the occurrence of a disease or disorder, within the necessary dosage and time period. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will typically be less than the therapeutically effective amount.

[0142] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will be the recipient of a specific treatment. The subject can be a human. The subject can be a patient suffering from a specific disease.

[0143] 7.2 Specific antibodies to internalizing receptors expressed on microglia, oligodendrocytes, and astrocytes

[0144] As described above, the present invention provides methods and uses of bispecific antibodies or antigen-binding fragments thereof, which, in addition to binding to Aβ proteins, also specifically bind to internalized receptors and / or antigens expressed on microglia, such as CD22, CD33, and CD74. As used herein, the term "antibody" and its grammatical equivalents refer to immunoglobulin molecules that recognize and specifically bind to targets (such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or any combination of the above substances) through at least one antigen binding site (wherein the antigen binding site is generally located within the variable region of the immunoglobulin molecule). As used herein, the term includes complete polyclonal antibodies, complete monoclonal antibodies, single domain antibodies (sdAb; e.g., camelid antibodies, alpaca antibodies), single chain Fv (scFv) antibodies, heavy chain antibodies (HCAb), light chain antibodies (LCAb), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecules (e.g., dual variable domain immunoglobulin molecules) comprising antigen binding sites, as long as these antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, rabbit antibodies, camel antibodies, primate antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), respectively referred to as α, δ, ε, γ, and μ based on the properties of their heavy chain constant domains. In some embodiments, an antibody may comprise four polypeptide chains, two heavy chains (H) and two light chains (L), which are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.

[0145] Unless expressly stated otherwise, as used herein, the term "antibody" includes "antigen-binding fragments" of intact antibodies. As used herein, the term "antigen-binding fragment" refers to a portion or fragment of an intact antibody that is the antigen-determining variable region of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains without a hinge region), Fab' (a monovalent fragment consisting of the VL, VH, CL, and CH1 domains attached to a hinge region), F(ab')2 (a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region), Fd (a fragment consisting of the VH and CH1 domains), Fv (a fragment consisting of the VL and VH domains of a single arm of an antibody), linear antibodies, single-chain antibody molecules (e.g., scFv, which is a single polypeptide chain with VL and VH regions joined by recombinant means), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide bond-linked scFv (dsscFv), diantibodies (bivalent, bispecific antibodies), triantibodies, tetraantibodies, miniantibodies, dual variable domain antibodies (DVD), single variable domain antibodies (sdAb or dAb; for example: camelid antibodies, alpaca antibodies), single variable domains of heavy chain antibodies (VHH), and bispecific or multispecific antibodies formed by antibody fragments. "Bispecific" antibodies are artificial hybrid antibodies with two different antigen binding sites that recognize and specifically bind to two different targets. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or connection of Fab' fragments. For example, see Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992); Labrijn AF et al., Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov. 18(8):585-608 (2019); Nie S et al., Biology drives the discovery of bispecific antibodies as innovative therapeutics. Antib Ther. 17; 3(1):18-62 (2020).

[0146] Antibodies or their antigen binding fragments can be part of a larger immunoadhesion molecule, formed by covalent or non-covalent binding of antibodies or antigen binding fragments to one or more other proteins or peptides. Examples of such immunoadhesion molecules include using streptavidin core regions to make tetrameric scFv molecules (Kipriyanov et al., (1995) HumanantibodiesandHybridomas6:93-101) and using cysteine ​​residues, labeled peptides and C-terminal polyhistidine tags to make bivalent and biotinylated scFv molecules (Kipriyanov et al., (1994) Mol.Immunol.31:1047-1058). Antigen binding fragments, such as Fab, Fab' and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as digesting whole antibodies with papain or pepsin, respectively. In addition, as described herein, antibodies, antigen binding fragments and immunoadhesion molecules can be obtained by standard DNA recombinant techniques.

[0147] The term "humanized antibody" as used herein refers to a form of non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human sequences. Humanized antibodies are typically human immunoglobulins. In some cases, residues in the Fv framework region of a human immunoglobulin are replaced by corresponding residues in an antibody of a non-human species. In some cases, residues in a CDR are replaced by residues in a CDR of a non-human species (e.g., mouse, rat, hamster, camel) having the desired specificity, affinity, and / or binding ability. Humanized antibodies can be further modified by replacing other residues in the Fv framework region and / or within the substituted non-human residues to improve and optimize the specificity, affinity, and / or binding ability of the antibody. The term "human antibody" as used herein refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any known technique in the art.

[0148] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion includes a variable region (VH) of about 120-130 or more amino acids and a carboxyl terminal portion including a constant region. In some embodiments, the heavy chain constant region consists of three domains, CH1, CH2, and CH3, and there is a short flexible hinge region connecting the CH1 and CH2 domains. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, respectively called alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Different heavy chains have different sizes: α, δ, and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, these different types of heavy chains produce five well-known classes of antibodies, IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4. The heavy chain may be a human heavy chain.

[0149] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxyl terminal portion including a constant region. The light chain constant region consists of a domain, CL. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant region, there are two different types, i.e., kappa (κ) and lambda (λ). The light chain amino acid sequence is well known in the art. The light chain can be a human light chain.

[0150] The term "variable domain" or "variable region" refers to a part of an antibody light chain or heavy chain, usually located at the amino terminus of the light chain or heavy chain, and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable domains vary greatly in sequence between different antibodies. The variability of the sequence is mainly concentrated in the CDR, and the less variable part of the variable domain is called the framework region (FR). The CDRs of the light and heavy chains are mainly responsible for the interaction between the antibody and the antigen. In some embodiments, each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid position numbering used herein is according to the EU index as in Kabat et al., (1991) Sequences of proteins of immunological interest. (U.S. Department of Health and Human Services, Washington, DC) 5th edition.

[0151] CDR refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the VHβ sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the VLβ sheet framework of an antibody. Therefore, CDR is a variable region sequence interspersed in the framework region sequence. CDR regions are well known to those skilled in the art and have been defined by a variety of methods / systems. These systems and / or definitions have been developed and improved for many years and include Kabat, Chothia, IMGT, ABM and Contact. For example, Kabat defines the region with the highest variability in the variable (V) domain of an antibody (Kabat et al., J.Biol.Chem.252:6609-6616 (1977); Kabat, Adv.Prot.Chem.32:1-75 (1978)). The Chothia definition is based on the position of the structural loop region, which defines the CDR region sequence as those residues that are not part of the conserved β-sheet framework and are therefore able to adapt to different conformations (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Both terms are recognized in the art. In addition, the IMGT system is based on sequence variability and position in the variable region structure. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. Software programs (e.g., abYsis) are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs. The position of CDRs within the canonical antibody variable domain has been determined by comparing a large number of structures (Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997); Morea et al., Methods 20: 267-279 (2000)). Because the number of residues in the hypervariable regions varies among different antibodies, additional residues relative to the canonical positions are often numbered next to the residue number using a, b, c, etc. in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). This nomenclature is also well known to those skilled in the art.

[0152] For example, the table below lists CDRs defined according to Kabat (hypervariable) or Chothia (structural) nomenclature.

[0153] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> Ring position VHCDR 31-35 26-32 Connecting chains B and C VHCDR2 50-65 53-55 Connecting C' and C" chains VHCDR3 95-102 96-101 Connecting F and G strands VLCDR 24-34 26-32 Connecting chains B and C VLCDR2 50-56 50-52 Connecting C' and C" chains VLCDR3 89-97 91-96 Connecting F and G strands

[0154] 1 The residue numbering follows the nomenclature of Kabat et al., supra.

[0155] 2 The residue numbering follows the nomenclature of Chothia et al., supra.

[0156] One or more CDRs can also be incorporated into a molecule in a covalent or non-covalent manner to make it an immunoadhesin. An immunoadhesin can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) non-covalently. The CDRs allow the immunoadhesin to bind to a specific target antigen.

[0157] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the site where an antibody or antigen binding fragment binds to the surface of a target molecule, such as a localized region on the surface of an antigen. The target molecule may include a protein, peptide, nucleic acid, carbohydrate, or lipid. An epitope with immunogenic activity is a portion of a target molecule that causes an immune response in an animal. An epitope of a target molecule with antigenic activity is a portion of a target molecule to which an antibody binds, which can be determined by any method known in the art (including, for example, by immunoassay). An antigenic epitope is not necessarily immunogenic. An epitope is typically composed of chemically active surface groups of a molecule such as an amino acid or a sugar side chain, and has specific three-dimensional structural characteristics and specific charge characteristics. The term "epitope" includes linear epitopes and conformational epitopes. The region in a target molecule (e.g., a polypeptide) that acts on an epitope may be a continuous amino acid of a polypeptide, or an epitope may be composed of a combination of two or more non-continuous regions of a target molecule. An epitope may or may not be a three-dimensional surface feature of a target molecule. Epitopes formed by contiguous amino acids (also called linear epitopes) are usually retained when the protein is denatured, while epitopes formed by tertiary folding (also called conformational epitopes) are usually lost when the protein is denatured. Epitopes usually include at least 3, and more commonly, at least 5, 6, 7 or 8-10 amino acids in a unique spatial conformation.

[0158] As used herein, the term "specifically binds" refers to a polypeptide or molecule that interacts with an epitope, protein or target molecule more frequently, more rapidly, longer lastingly, more strongly with affinity than an alternative substance (including related or unrelated proteins), or with some combination thereof. The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by methods such as immunoassay, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore) or other techniques known to those skilled in the art. Typically, the specific reaction is at least twice the background signal or noise, and can be more than 10 times the background. For discussion of antibody specificity, see Fundamental Immunology Second Edition 1989, edited by Paul, Raven Press, New York, pp. 332-336. The binding moiety that specifically binds to a target molecule can bind to a target molecule with an affinity higher than its affinity for different molecules. In some embodiments, a binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity that is at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher than its affinity for a different molecule. In some embodiments, a binding moiety that specifically binds to a particular target molecule binds to the different molecule with such low affinity that binding cannot be detected using an assay described herein or known in the art. In some embodiments, "specifically binds" means, for example, that the binding moiety has a K of about 0.1 mM or less. D In some embodiments, "specifically binds" refers to a polypeptide or molecule that binds to a target molecule with a K of about 10 μM or less or about 1 μM or less. D In some embodiments, "specifically binds" refers to a polypeptide or molecule that binds to a target with a K of about 0.1 μM or less, about 0.01 μM or less, about 1 nM or less. DBinding to a target. Due to the sequence identity between homologous proteins of different species, specific binding may include polypeptides or molecules that recognize proteins or targets of more than one species. Similarly, due to the homology in certain regions of the polypeptide sequences of different proteins, specific binding may include polypeptides or molecules that recognize more than one protein or target. It is understood that, in some embodiments, a binding portion (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding, i.e., binding to a single target. Therefore, in some embodiments, a binding portion (e.g., an antibody) may specifically bind to more than one target. For example, in some cases, an antibody may include two identical antigen binding sites, each of which can specifically bind to the same epitope on two or more proteins. In some alternative embodiments, an antibody may be bispecific and include at least two antigen binding sites with different specificities.

[0159] As used herein, the term "binding affinity" generally refers to the sum of the strengths of non-covalent interactions between a binding moiety and a target molecule (e.g., an antigen). The binding of a binding moiety to a target molecule is a reversible process, and the binding affinity is usually expressed in terms of the equilibrium dissociation constant (K D ) to report. D is the dissociation rate (k off or k d ) and the association rate (k on or k a ) ratio. D The lower the K, the higher the affinity. A is the equilibrium association constant, which is also the reciprocal of the equilibrium dissociation constant, i.e. = 1 / K D For antibody-antigen interactions, K D It can be calculated as the ratio of the product of the free antibody and free antigen concentrations to the antibody-antigen complex concentration, that is, [antigen] x [antibody] / [antigen-antibody].

[0160] There are many methods known in the art for measuring binding affinity, any of which may be used for the purposes of the present disclosure. Specific illustrative embodiments include the following. In some embodiments, "K D ” or “K D The "KD value" can be measured by assays known in the art, such as by binding assays. D Can be measured in a radiolabeled antigen binding assay (RIA) (Chen et al., (1999) J. Mol Biol 293:865-881). Dor K D K values ​​can also be measured by biolayer interferometry (BLI), for example using the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K D Values ​​can also be measured by surface plasmon resonance assay using a BIAcore system (eg, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

[0161] As used herein, the term "variant" in relation to a protein or polypeptide having a specific sequence feature ("reference protein" or "reference polypeptide") refers to a different protein or polypeptide having one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions compared to the reference protein or reference polypeptide. The change in the amino acid sequence may be an amino acid substitution. The change in the amino acid sequence may be a conservative amino acid substitution. A functional fragment or functional variant of a protein or polypeptide retains the basic structural and functional properties of the reference protein or polypeptide.

[0162] As used interchangeably herein, the terms "polypeptide", "peptide", "protein" and their grammatical equivalents refer to an amino acid polymer of any length, which may be linear or branched. It may include non-natural or modified amino acids, or may be interrupted by non-amino acids. The polypeptide, peptide or protein may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification.

[0163] As used interchangeably herein, the terms "polynucleotide", "nucleic acid" and their grammatical equivalents refer to nucleotide polymers of any length, and include DNA and RNA. Nucleotides can be deoxynucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Nucleic acid molecules can be single-stranded or double-stranded.

[0164] As used herein, the term "encode" and its grammatical equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid (such as a gene, cDNA or mRNA), which serves as a template for synthesizing other polymers and macromolecules in a biological process, and these polymers and macromolecules have a definite nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a definite amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to the gene produce a protein, the gene encodes a protein. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The nucleotide sequence encoding a protein and RNA may include introns.

[0165] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition refers to a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition that is not present in a form found in nature. An isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition includes those that have been purified to the point where they no longer exist in a form found in nature. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is substantially pure. In some embodiments, an isolated polypeptide, peptide, protein, antibody, polynucleotide, vector, cell, or composition is substantially free of other cellular material and / or chemicals.

[0166] As used herein, the term "identical", percentage "identity" and grammatical equivalents thereof used when relating to two or more polynucleotides or polypeptides, refers to two or more sequences or subsequences when comparing and aligning (if necessary, introducing spaces) to achieve maximum correspondence, without considering any conservative amino acid substitutions as part of sequence identity, the nucleotides or amino acid residues that are identical or have a specific percentage are identical. Percent identity can be measured using sequence alignment software or algorithms or by visual estimation. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variants thereof. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, which means that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleotide or amino acid residue identity when using a sequence comparison algorithm or by visual inspection to achieve maximum correspondence. In some embodiments, identity exists in a region of an amino acid sequence having a length of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists in a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequence is substantially identical over the full length of the compared sequence (such as the coding region of a target protein or antibody). In some embodiments, the identity exists in a region of the nucleotide sequence that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, the identity exists in a region that is longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the full length of the compared sequences (such as the nucleotide sequence encoding the target protein).

[0167] As used herein, "conservative amino acid substitution" refers to the replacement of an amino acid residue by another amino acid residue having a similar side chain. As used herein, "conservatively similar" amino acids or residues refer to non-identical amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0168] As used herein, the term "vector" and its grammatical equivalents refer to a vector for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and / or expressed in the host cell. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or marker that can be operably stably integrated into the host cell chromosome. In addition, the vector may also include one or more selectable marker genes and appropriate expression control sequences. For example, the selectable marker gene that may be included provides resistance to antibiotics or toxins, supplements nutritional deficiency defects, or supplies key nutrients that are not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., which are all well-known in the art. When two or more polynucleotides are to be co-expressed, the two polynucleotides may be inserted into, for example, a single expression vector or a separate expression vector. For single vector expression, the encoding polynucleotide may be operably connected to a common expression control sequence or connected to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into a host cell may be confirmed using methods well-known in the art. Those skilled in the art understand that the polynucleotides are expressed in sufficient amounts to produce the desired product (e.g., an anti-CD22, anti-CD33, or anti-CD74 bispecific antibody or antigen-binding fragment that simultaneously targets Aβ protein), and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0169] As used herein, the term "host cell" refers to a cell into which genetic material (such as a recombinant expression vector) may be or has been introduced. Host cells include not only subject cells into which exogenous genetic material has been introduced, but also the offspring of such cells. Due to mutations or environmental influences, offspring cells may undergo certain modifications, so these offspring may not be identical to parental cells.

[0170] As used herein and understood in the art, "EC" refers to the effective concentration of an agent (e.g., an antibody), and is commonly used in dose-response curves. The "effect" of a formulation can be a positive (activation) effect or a negative effect. The term "EC50" refers to the concentration at which an active agent (e.g., an antibody) produces a half-maximal response. Also as used herein and understood in the art, "IC" refers to the concentration of an agent with an inhibitory effect, and is also commonly used in dose-response curves. The term "IC50" refers to the concentration of an agent (e.g., an antibody), at which the activity of its inhibition is reduced by half.

[0171] 7.2.1 Exemplary Anti-CD22, Anti-CD33 and Anti-CD74 Bispecific Antibodies

[0172] As described in the previous section, provided herein are methods and uses of bispecific antibodies or antigen-binding fragments thereof, which, in addition to binding to Aβ protein, also specifically bind to CD22, CD33 or CD74, which (a) promotes the cis-trans conversion of CD22 or CD33; and / or (b) induces the internalization of CD22, CD33 and CD74. The receptor binding portions of the bispecific antibodies and antigen-binding fragments provided herein can be used to clear Aβ, reduce neuroinflammation, or both. The bispecific antibodies and antigen-binding fragments provided herein can also be used to treat diseases or disorders associated with Aβ and / or neuroinflammation. In some embodiments, the bispecific antibodies used in the methods provided herein specifically bind to human CD22. In some embodiments, the bispecific antibodies used in the methods provided herein specifically bind to human CD33. In some embodiments, the bispecific antibodies used in the methods provided herein specifically bind to human CD74.

[0173] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies that can be used in the methods disclosed herein are IgA, IgD, IgE, IgG or IgM antibodies. In some embodiments, the bispecific antibody is an IgA antibody. In some embodiments, the bispecific antibody is an IgD antibody. In some embodiments, the bispecific antibody is an IgE antibody. In some embodiments, the bispecific antibody is an IgG antibody. In some embodiments, the bispecific antibody is an IgM antibody. In some embodiments, the bispecific antibody provided herein can be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody. In some embodiments, the bispecific antibody is an IgG1 antibody. In some embodiments, the bispecific antibody is an IgG2 antibody. In some embodiments, the bispecific antibody is an IgG3 antibody. In some embodiments, the bispecific antibody is an IgG4 antibody. In some embodiments, the bispecific antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, or any one of the foregoing constant regions, whose glycosylation sites and / or glycoforms at the glycosylation sites are modified.

[0174] In some embodiments, the antigen binding fragment of an anti-CD22 antibody, an anti-CD33 antibody, or an anti-CD74 antibody is used in the methods disclosed herein. In some embodiments, the antigen binding fragment provided herein can be a single domain antibody (sdAb), a heavy chain antibody (HCAb), Fab, Fab', F(ab')2, Fv, a single chain variable fragment (scFv) or (scFv)2. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33, or anti-CD74 antibody is a single domain antibody (sdAb). In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33, or anti-CD74 antibody is a heavy chain antibody (HCAb). In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33, or anti-CD74 antibody is a Fab. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33, or anti-CD74 antibody is a Fab'. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33, or anti-CD74 antibody is a F(ab')2. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33 or anti-CD74 antibody is an Fv. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33 or anti-CD74 antibody is an scFv. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33 or anti-CD74 antibody is a disulfide-linked scFv [(scFv)2]. In some embodiments, the antigen binding fragment of an anti-CD22, anti-CD33 or anti-CD74 antibody is a diabody (dAb).

[0175] In some embodiments, the methods disclosed herein are recombinant anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include camelids (e.g., camels, dromedaries and llamas) antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include humanized antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein include human antibodies or antigen-binding fragments. In some embodiments, provided herein are anti-CD22, anti-CD33 or anti-CD74 humanized scFvs. In some embodiments, provided herein are anti-CD22, anti-CD33 or anti-CD74 humanized Fabs.

[0176] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods provided herein are isolated. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods provided herein are substantially pure.

[0177] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen binding fragments used in the methods provided herein comprise a monovalent antigen binding site. In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen binding fragments comprise a monospecific binding site. In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen binding fragments comprise a bivalent binding site.

[0178] In some embodiments, monoclonal antibodies are modified using recombinant DNA technology to produce alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of mouse monoclonal antibodies are replaced by the constant regions of human antibodies to generate chimeric antibodies. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragments of monoclonal antibodies. In some embodiments, site-directed or high-density mutagenesis is performed on the variable regions to optimize the specificity and / or affinity of the monoclonal antibodies.

[0179] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein are humanized antibodies or antigen-binding fragments. Various methods for generating humanized antibodies are known in the art. Methods known in the art can achieve high affinity binding of humanized antibodies. A non-limiting example of this method is hypermutation of the variable region and selection of cells expressing such high affinity antibodies (affinity maturation). In addition to using display libraries, specified antigens (e.g., epitopes of recombinant CD22, recombinant CD33, recombinant CD74, or their respective recombinant proteins) can also be used to immunize non-human animals, such as rodents. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be produced and separated using methods known in the art and / or methods disclosed herein. In some embodiments, mice can be immunized with antigens (e.g., epitopes of recombinant CD22, recombinant CD33, recombinant CD74, or their respective recombinant proteins).

[0180] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein are human antibodies or antigen-binding fragments. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced by immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced by lymphocytes isolated from immune individuals. In any case, cells that produce antibodies against targeted antigens can be produced and isolated. In some embodiments, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies. Alternatively, phage display technology can also be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene libraries of unimmunized donors. The generation and use of antibody phage libraries are well known in the art. Once the antibody is determined, affinity maturation strategies known in the art can be used, including but not limited to chain shuffling and site-directed mutagenesis, to produce human antibodies with higher affinity. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. Following immunization, these mice are able to produce a full human antibody repertoire in the absence of endogenous immunoglobulin production.

[0181] The specific CDR sequences defined herein are generally based on a combination of the Kabat and Chothia definitions. However, it is understood that reference to a heavy chain CDR or multiple heavy chain CDRs and / or a light chain CDR or multiple light chain CDRs of a specific antibody includes all CDR definitions known to those of ordinary skill in the art. Anti-CD22 antibodies or antigen-binding fragments that can be used in the methods herein include SM03 and SM06 and variants and derivatives thereof.

[0182] The term "SM03" refers to a chimeric antibody against human CD22 (hCD22). The sequence characteristics of SM03 are shown in the table below. For more description of the structural and functional characteristics of SM03, see, for example, Yang et al., (2006), Chinese J New Drug 15(3):186-92; and Chinese Patent No. ZL03123054.7, the entire contents of which are incorporated herein by reference.

[0183] The term "SM06" refers to a framework-patched or humanized version of the redesigned chimeric antibody SM03 to reduce its potential immunogenicity and have affinity and specificity for hCD22. The sequence characteristics of SM06 are shown in the table below. For more description of the structural and functional characteristics of SM06, see, for example, Liang et al., (2006) Chinese J New Drug 15(21): 1832-36; Chinese Patent No. ZL 01144894.6, and U.S. Patent Nos. 7,321,029B2 and 7,338,659B2, the entire contents of which are incorporated herein by reference.

[0184] Table 1: CDR sequences of SM03 and SM06

[0185]

[0186] Table 2: Variable regions of SM03 and SM06

[0187]

[0188] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of SM03 / SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs in Table 1. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs in Table 1. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs and one, two and / or three VH CDRs in Table 1.

[0189] The term "LL2" refers to a chimeric antibody against human CD22 (hCD22). The sequence characteristics of LL2 are shown in the table below. For more description of the structural and functional characteristics of LL2, see Leung et al. (1994) Chimerization of LL2, a rapidly internalizing antibody specific for B-cell lymphoma. Hybridoma 13: 469-476; Stein R, et al., (1993) Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody, LL2. Cancer Immunol Immunother; 37(5): 293-8.; U.S. Patent No. 5,789,554A, the entire contents of which are incorporated herein by reference. The term "hLL2" refers to a framework-patched or humanized version of the redesigned chimeric antibody LL2 to reduce its potential immunogenicity and exhibit affinity and specificity for hCD22. The sequence characteristics of hLL2 are shown in the table below. For more description of the structural and functional characteristics of hLL2, see, for example, Leung et al., (1995) Construction and characterization of a humanized, internalizing B-cell (CD22)-specific, leukemia / lymphoma antibody, LL2. Mol. Immunol. 32: 1413-1427; Juweid M et al., (1995) Treatment of non-Hodgkin's lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody. Cancer Res. 55(23 Suppl): 5899s-5907s.; U.S. Pat. No. 5,789,554A, the entire contents of which are incorporated herein by reference.

[0190] Table 3: CDR sequences of LL2 and hLL2

[0191]

[0192] Table 4: Variable regions of LL2 and hLL2

[0193]

[0194] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of LL2 / hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs in Table 3. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs in Table 3. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein include one, two and / or three VLCDRs in Table 3 and one, two and / or three VH CDRs.

[0195] The term "Gemtuzumab" refers to a framework-repaired or humanized version of a redesigned anti-CD33 antibody to reduce its potential immunogenicity and exhibit affinity and specificity for hCD33. The sequence characteristics of Gemtuzumab are shown in the table below. For more description of the structural and functional characteristics of Gemtuzumab, see, for example, Bernstein ID (2000). Monoclonal antibodies to the myeloid stem cells: therapeutic implications of CMA-676, a humanized anti-CD33 antibody calicheamicin conjugate. Leukemia. 14 (3): 474-5.; U.S. Pat. No. 5,877,296A, the entire contents of which are incorporated herein by reference.

[0196] Table 5: CDR sequences of gemtuzumab

[0197]

[0198] Table 6: Variable regions of gemtuzumab

[0199]

[0200] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs of Table 5. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs of Table 5. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs of Table 5 and one, two and / or three VH CDRs.

[0201] The term "HuMy9-6" refers to a framework-patched or humanized version of a redesigned anti-CD33 antibody to reduce its potential immunogenicity and exhibit affinity and specificity for hCD33. The sequence characteristics of HuMy9-6 are shown in the table below. For more descriptions of the structural and functional characteristics of HuMy9-6, see, for example, Lapusan et al., (2012) Phase I studies of AVE9633, an anti-CD33 antibody-maytansinoid conjugate, in adult patients with relapsed / refractory acute myeloid leukemia. Invest New Drugs. 30(3): 1121-31.; U.S. Pat. No. 10,000,566B2, the entire contents of which are incorporated herein by reference.

[0202] Table 7: CDR sequences of HuMy9-6

[0203]

[0204] Table 8: Variable regions of HuMy9-6

[0205]

[0206] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of HuMy9-6. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs of Table 7. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs of Table 7. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include one, two and / or three VLCDRs of Table 7 and one, two and / or three VH CDRs.

[0207] The term "Lintuzumab" refers to a framework-patched or humanized version of a redesigned anti-CD33 antibody to reduce its potential immunogenicity and exhibit affinity and specificity for hCD33. The sequence characteristics of lintuzumab are shown in the table below. For more description of the structural and functional characteristics of lintuzumab, see, for example, Gibson AD. (2002) Phase III trial of a humanized anti-CD33 antibody (HuM195) in patients with relapsed or refractory acute myeloid leukemia. Clin Lymphoma. 3 (1): 18-9.; U.S. Pat. No. 9,061,074 B2, the entire contents of which are incorporated herein by reference.

[0208] Table 9: CDR sequences of lintuzumab

[0209]

[0210] Table 10: Variable regions of lintuzumab

[0211]

[0212] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs of Table 9. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs of Table 9. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs and one, two and / or three VH CDRs of Table 9.

[0213] The term "cLL1" refers to a chimeric antibody against human CD74 (hCD74). The sequence characteristics of LL1 are shown in the table below. For more description of the structural and functional characteristics of LL1, see, for example, Stein R et al., (2007) CD74: a new candidate target for the immunotherapy of B-cell neoplasms. Clin Cancer Res. 13 (18 Pt 2): 5556s-5563s.; U.S. Pat. No. 8,119,101B2, the entire contents of which are incorporated herein by reference.

[0214] The term "hLL1" refers to a framework-patched or humanized version of a redesigned anti-CD74 antibody to reduce its potential immunogenicity and exhibit affinity and specificity for hCD74. The sequence characteristics of hLL1 are shown in the table below. For more description of the structural and functional characteristics of hLL1, see, for example, Stein R et al. (2007) CD74: a new candidate target for the immunotherapy of B-cell neoplasms. Clin Cancer Res. 13 (18 Pt 2): 5556s-5563s.; U.S. Pat. No. 8,119,101B2, the entire contents of which are incorporated herein by reference.

[0215] Table 11: CDR sequences of cLL1 / hLL1

[0216]

[0217] Table 12: Variable regions of cLL1 / hLL1

[0218]

[0219] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five and / or six CDRs of cLL1 / hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs of Table 11. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs of Table 11. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs of Table 11 and one, two and / or three VH CDRs.

[0220] The term "Aducanumab" refers to a framework-patched or humanized version of a redesigned anti-Aβ antibody to reduce its potential immunogenicity and exhibit affinity and specificity for Aβ. The sequence characteristics of Aducanumab are shown in the table below. For more description of the structural and functional characteristics of Aducanumab, see, for example, Sevigny J et al. (2016) The antibody aducanumab reduces Aβplaques in Alzheimer's disease. Nature. 537(7618): 50-6.; U.S. Pat. No. 9,670,272B2, the entire contents of which are incorporated herein by reference.

[0221] Table 13: CDR sequences of aducanumab

[0222]

[0223] Table 14: Variable regions of aducanumab

[0224]

[0225] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five, and / or six CDRs of aducanumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments include a VL comprising one, two, and / or three VL CDRs in Table 13. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include a VH comprising one, two, and / or three VH CDRs in Table 13. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include one, two, and / or three VLCDRs of Table 13 and one, two, and / or three VH CDRs.

[0226] The term "BAN2401" refers to a framework-repaired or humanized version of a redesigned anti-Aβ antibody to reduce its potential immunogenicity and exhibit affinity and specificity for Aβ. The sequence characteristics of BAN2401 are shown in the table below. For more description of the structural and functional characteristics of BAN2401, see, for example, Tucker S et al. (2015) The murine version of BAN2401 (mAb158) selectively reduces amyloid-β protofibrils in brain and cerebrospinal fluid of tg-ArcSwe mice. J Alzheimers Dis. 43 (2): 575-88.; U.S. Pat. No. 8,025,878B2, the entire contents of which are incorporated herein by reference.

[0227] Table 15: CDR sequences of BAN2401

[0228]

[0229] Table 16: Variable regions of BAN2401

[0230]

[0231] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments useful for the methods provided herein include one, two, three, four, five and / or six CDRs of BAN2401. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs in Table 15. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs in Table 15. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs and one, two and / or three VH CDRs in Table 15.

[0232] The term "Gantenerumab" refers to a framework-repaired or humanized version of a redesigned anti-Aβ antibody to reduce its potential immunogenicity and exhibit affinity and specificity for Aβ. The sequence characteristics of Gantenerumab are shown in the table below. For more description of the structural and functional characteristics of Gantenerumab, see, for example, Bohrmann B et al. (2012) Gantenerumab: a novel human anti-Aβantibody demonstrates sustained cerebral amyloid-βbinding and elicits cell-mediated removal of human amyloid-β. J Alzheimers Dis. 28 (1): 49-69.; U.S. Pat. No. 7,794,719B2, the entire contents of which are incorporated herein by reference.

[0233] Table 17: CDR sequences of gantnerumab

[0234]

[0235] Table 18: Variable regions of gantnerumab

[0236]

[0237] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five, and / or six CDRs of gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments include a VL comprising one, two, and / or three VL CDRs of Table 17. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include a VH comprising one, two, and / or three VH CDRs of Table 17. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include one, two, and / or three VLCDRs of Table 17 and one, two, and / or three VH CDRs.

[0238] The term "Crenezumab" refers to a framework-repaired or humanized version of a redesigned anti-Aβ antibody to reduce its potential immunogenicity and exhibit affinity and specificity for Aβ. The sequence characteristics of Crenezumab are shown in the table below. For more description of the structural and functional characteristics of Crenezumab, see, for example, Bohrmann B et al. (2012) Gantenerumab: a novel human anti-Aβantibody demonstrates sustained cerebral amyloid-βbinding and elicits cell-mediated removal of human amyloid-β. J Alzheimers Dis. 28(1): 49-69.; U.S. Pat. No. 7,794,719B2, the entire contents of which are incorporated herein by reference.

[0239] Table 19: CDR sequences of cranizumab

[0240]

[0241] Table 20: Variable regions of cranizumab

[0242]

[0243] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments useful in the methods provided herein include one, two, three, four, five, and / or six CDRs of cranelituzumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments include a VL comprising one, two, and / or three VL CDRs of Table 19. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include a VH comprising one, two, and / or three VH CDRs of Table 19. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include one, two, and / or three VLCDRs of Table 19 and one, two, and / or three VH CDRs.

[0244] The term "AD38" refers to a framework-repaired or humanized version of a redesigned anti-Aβ antibody to reduce its potential immunogenicity and exhibit affinity and specificity for Aβ. The sequence characteristics of AD38 are shown in the table below. For more description of the structural and functional characteristics of AD38, see, for example, WO Patent No. WO2006,103,116A1, the entire contents of which are incorporated herein by reference.

[0245] Table 21: CDR sequences of AD38

[0246]

[0247] Table 22: Variable regions of AD38

[0248]

[0249] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments useful for the methods provided herein include one, two, three, four, five and / or six CDRs of AD38. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments include a VL comprising one, two and / or three VL CDRs in Table 21. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include a VH comprising one, two and / or three VH CDRs in Table 21. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments provided herein include one, two and / or three VL CDRs and one, two and / or three VH CDRs in Table 21.

[0250] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 1; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence of SEQ ID NO: 2; or (3) a light chain CDR3 (VLCDR3) having an amino acid sequence of SEQ ID NO: 3; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD22 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 3; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0251] In some embodiments, the antibodies or antigen-binding fragments thereof that can be used in the methods provided herein include a light chain variable region (VL) that includes (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 11; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence of SEQ ID NO: 12; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD22 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 11; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 12; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0252] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 21; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 22; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 23; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD33 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDR. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDR.

[0253] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 29; (2) a light chain CDR2 (VL CDR2) having an amino acid sequence of SEQ ID NO: 30; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 31; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD33 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 29; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 30; (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 31; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acids in the VL CDR. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDR.

[0254] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 37; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 38; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 39; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD33 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 37; (2) a VLCDR2 having an amino acid sequence of SEQ ID NO: 38; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 39; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDR. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDR.

[0255] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 45; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 46; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 47; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to CD74 comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 45; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 46; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 47; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0256] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 55; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 56; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 57; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Aβ comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 55; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 56; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 57; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0257] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR (VL CDR1) having an amino acid sequence of SEQ ID NO: 64; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 65; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 66; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Aβ comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 64; (2) a VL CDR3 having an amino acid sequence of SEQ ID NO: 65; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 66; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDR. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDR.

[0258] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 72; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 73; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 74; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Aβ comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 72; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 74; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0259] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 80; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 81; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 82; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Aβ comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 80; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 81; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 82; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDR. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDR.

[0260] In some embodiments, the antibodies or antigen-binding fragments thereof useful in the methods provided herein include a light chain variable region (VL) comprising (1) a light chain CDR1 (VL CDR1) having an amino acid sequence of SEQ ID NO: 88; (2) a light chain CDR (VL CDR2) having an amino acid sequence of SEQ ID NO: 89; or (3) a light chain CDR3 (VL CDR3) having an amino acid sequence of SEQ ID NO: 90; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to Aβ comprises a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 88; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 89; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 90; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VL CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VL CDRs.

[0261] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 4; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 5; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 6; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 5; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 6; or a variant thereof having a substitution, addition, and / or deletion of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDRs. In some embodiments, the variant has a substitution, addition, and / or deletion of up to about 5 amino acids in the VH CDRs.

[0262] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 14; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 15; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 16; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 15; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 16; or a variant thereof having a substitution, addition, and / or deletion of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition, and / or deletion of up to about 5 amino acids in the VH CDR.

[0263] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 24; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 25; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 26; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 25; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 26; or a variant thereof having a substitution, addition, and / or deletion of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition, and / or deletion of up to about 5 amino acids in the VH CDR.

[0264] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 32; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 33; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 34; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 32; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 33; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 34; or a variant thereof having a substitution, addition and / or deletion of up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0265] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 40; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 41; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 42; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 42; or a variant thereof having a substitution, addition and / or deletion of up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0266] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD74, comprising a heavy chain variable region (VH) comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 48; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 49; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 50; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD74, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 48; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 49; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 50; or a variant thereof having a substitution, addition, and / or deletion of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDRs. In some embodiments, the variant has a substitution, addition, and / or deletion of up to about 5 amino acids in the VH CDRs.

[0267] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a heavy chain variable region (VH), the heavy chain variable region comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 58; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 59; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 60; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 58; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 59; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 60; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDRs. In some embodiments, the variant has a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDRs.

[0268] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a heavy chain variable region (VH), the heavy chain variable region comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 67; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 68; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 69; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 67; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 68; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 69; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VH CDRs.

[0269] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a heavy chain variable region (VH), the heavy chain variable region comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 75; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 76; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 77; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 75; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 76; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 77; or a variant thereof having a substitution, addition, and / or deletion of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition, and / or deletion of up to about 5 amino acids in the VH CDR.

[0270] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a heavy chain variable region (VH), the heavy chain variable region comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 83; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 84; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 85; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 83; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 84; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 85; or a variant thereof having substitutions, additions and / or deletions of up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acids in the VH CDRs. In some embodiments, the variant has substitutions, additions and / or deletions of up to about 5 amino acids in the VH CDRs.

[0271] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a heavy chain variable region (VH), the heavy chain variable region comprising (1) a heavy chain CDR1 (VHCDR1) having an amino acid sequence of SEQ ID NO: 91; (2) a heavy chain CDR2 (VH CDR2) having an amino acid sequence of SEQ ID NO: 92; or (3) a heavy chain CDR3 (VH CDR3) having an amino acid sequence of SEQ ID NO: 93; or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has a maximum of about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 91; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 92; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 93; or a variant thereof having a substitution, addition and / or deletion of up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acids in the VH CDR. In some embodiments, the variant has a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0272] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 2; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 3; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 4; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 5; (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 6; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0273] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 11; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 12; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 13; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 14; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 15; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 16; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0274] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 21; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 22; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 23; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 24; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 25; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 26; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0275] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 29; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 30; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 31; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 32; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 33; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 34; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0276] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 37; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 38; and (3) a VL CDR3 having SEQ ID NO: 39; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 40; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 41; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 42; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0277] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 45; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 46; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 47; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 48; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 49; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 50; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0278] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 55; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 56; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 57; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR2 having an amino acid sequence of SEQ ID NO: 59; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 60; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0279] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 64; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 65; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 66; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 67; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 68; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 69; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0280] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 72; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 73; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 74; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 75; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 76; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 77; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0281] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 80; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 81; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 82; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 83; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 84; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 85; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0282] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising (1) a VL CDR1 having an amino acid sequence of SEQ ID NO: 88; (2) a VL CDR2 having an amino acid sequence of SEQ ID NO: 89; and (3) a VL CDR3 having an amino acid sequence of SEQ ID NO: 90; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VL CDR; and (b) a VH comprising (1) a VH CDR1 having an amino acid sequence of SEQ ID NO: 91; (2) a VH CDR2 having an amino acid sequence of SEQ ID NO: 92; and (3) a VH CDR3 having an amino acid sequence of SEQ ID NO: 93; or a variant thereof having a substitution, addition and / or deletion of up to about 5 amino acids in the VH CDR.

[0283] It is well known in the art that the VH CDR3 and VL CDR3 domains play an important role in the binding specificity / affinity of an antibody to an antigen. Therefore, in some embodiments, the anti-CD22, anti-CD33 and anti-CD74 bispecific antibodies or antigen-binding fragments thereof that can be used in the methods disclosed herein can have appropriate binding / dissociation kinetics with the corresponding receptor / antigen and have VH CDR3 and VL CDR3 that are identical or related to the corresponding antibody structure. For illustrative purposes, for example, taking the anti-CD22 antibody SM03 as an example, the common motif of the SM03 VL CDR3 including the amino acid sequence: QQGNTLPWT (SEQ ID NO: 3) can be modified by replacing one or more amino acids to adjust the affinity of the antibody without changing its binding specificity, or alternatively replaced with the VL CDR3 of an unrelated human antibody that is sufficiently similar to the SM03 VL CDR3, and the standard for use thereof is as described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Similarly, the common motif of SM03 VH CDR3 including the amino acid sequence: HSGYGSSYGVLFAY (SEQ ID NO: 6) can be modified by replacing one or more amino acids to adjust the affinity of the antibody without changing its binding specificity, or alternatively, the VH CDR3 of an unrelated human antibody with sufficient similarity to SM03 VH CDR3 can be substituted, using the standard described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Those skilled in the art will understand that certain substitutions of amino acids in the CDR3 domain will not change the epitope specificity of the antibody, especially substitutions with conservative amino acids.Therefore, in some embodiments, the CDR3 of the antibodies or antigen-binding fragments provided herein (e.g., SM03, SM06, LL2, hLL2, gemtuzumab, HuMy9-6, lintuzumab cLL1, hLL1, aducanumab, BAN2401, gantnerumab, cranizumab, AD38) can be replaced with a CDR3 from a human or primate antibody, which: (1) has the same number of residues as the CDR3 of the corresponding antibody and exhibits 50% or more sequence homology; (2) contains at least one, preferably multiple, residues that are identical to those in the CDR3 of the corresponding antibody; (3) contains at least one, preferably multiple, charged residues that are identical or conservatively similar to the residues at the corresponding positions in the CDR3 of the corresponding antibody; and / or (4) contains at least one, preferably multiple, amino acid residues that are identical or conservatively similar to the residues at the corresponding positions in the CDR3 of the corresponding antibody, and the position is known to be very important for maintaining the binding site structure / contact of the corresponding antibody, as determined by crystal structure and / or computer database analysis (e.g., see Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference). In some embodiments, no more than one to five conservative amino acid substitutions are made to the VL and / or VH CDR3 domains, or the VL and / or VH CDR3 of the corresponding antibody is replaced with the VL and / or VH CDR3 of the corresponding antibody of an unrelated primate or human antibody containing no more than one to five conservatively similar residues. In some embodiments, no more than one to three conservative amino acid substitutions are made within the VL and / or VH CDR3 domains, or the VL and / or VH CDR3 of a corresponding antibody is replaced with the VL and / or VH CDR3 of a corresponding antibody from an unrelated primate or human antibody that contains no more than one to three conservatively similar residues.

[0284] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 7 and NO: 8, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 9 and NO: 10, respectively.

[0285] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 7 and NO: 18, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and NO: 20, respectively.

[0286] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 27 and NO: 28, respectively.

[0287] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 35 and NO: 36, respectively.

[0288] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 43 and NO: 44, respectively.

[0289] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD74, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 51 and NO: 52, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 53 and NO: 54, respectively.

[0290] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 61 and NO: 62, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 61 and NO: 63, respectively.

[0291] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 70 and NO: 71, respectively.

[0292] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 78 and NO: 79, respectively.

[0293] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, which comprises VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 86 and NO: 87, respectively.

[0294] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, which comprises VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 94 and NO: 95, respectively.

[0295] Anti-CD22, anti-CD33, anti-CD74 and anti-Aβ antibodies or antigen-binding fragments thereof may include any VL disclosed herein and any VH disclosed herein in combination. In some embodiments, VL and VH are connected by a linker. The linker may be a flexible linker or a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker has an amino acid sequence of (GGGGS)n, n=3, 4 or 5 (SEQ ID NO: 142). In some embodiments, the linker has an amino acid sequence of GSAGSAAGSGEF (SEQ ID NO: 161). In some embodiments, the linker has an amino acid sequence of KESGSVSSEQLAQFRSLD (SEQ ID NO: 162). In some embodiments, the linker has an amino acid sequence of EGKSSGSGSESKS (SEQ ID NO: 163). In some embodiments, the linker has an amino acid sequence of SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 164). In some embodiments, the linker has an amino acid sequence of GGGGG (SEQ ID NO: 165). In some embodiments, the linker is a rigid linker. In some embodiments, the linker has an amino acid sequence of (EAAAK)n, n=3, 4 or 5 (SEQ ID NO: 143). In some embodiments, the linker has an amino acid sequence of (PA)nP, n=1, 2, 3, 4 or 5 (SEQ ID NO: 144 and SEQ ID NOs: 166-168).

[0296] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD22, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 7 or 9; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 8 or 10. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD22, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 9; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 10.

[0297] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD22, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 17 or 19; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 18 or 20. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD22, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 19; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 20.

[0298] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 27; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 28.

[0299] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 35; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 36.

[0300] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD33, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 43; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 44.

[0301] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD74, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 51 or 53; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 52 or 54. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD74, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 53; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 54.

[0302] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 61; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 62 or 63. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 61; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 63.

[0303] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 70; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 71.

[0304] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 78; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 79.

[0305] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 86; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 87.

[0306] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to Aβ, comprising (a) a VL comprising VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 94; and / or (b) a VH comprising VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 95.

[0307] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to CD22, comprising a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 7. In some embodiments, the VL comprises VL CDRs 1, 2, and 3 from a VL having an amino acid sequence of SEQ ID NO: 9.

[0308] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 8. In some embodiments, the VH comprises VH CDRs 1, 2, and 3 from a VH having an amino acid sequence of SEQ ID NO: 10.

[0309] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL and a VH, wherein the VL comprises a VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence of SEQ ID NO: 7, and the VH comprises a VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence of SEQ ID NO: 8. In some embodiments, the VL comprises a VL CDR1, CDR2, and CDR3 from a VL having an amino acid sequence of SEQ ID NO: 9, and the VH comprises a VH CDR1, CDR2, and CDR3 from a VH having an amino acid sequence of SEQ ID NO: 10.

[0310] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as SM03. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have VL from SM03. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have VH from SM03. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have VL and VH from SM03 at the same time. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have VL including VL CDR 1, 2 and 3 of the VL from SM03. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have VH including VHCDR 1, 2 and 3 of the VH from SM03. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have VL including VL CDR 1, 2 and 3 and VH including VH CDR 1, 2 and 3, which are respectively from the VL and VH of SM03. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are variants of SM03. The VL of the SM03 variant can be a variant of the VL of SM03 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:7. The VH of the SM03 variant can be a variant of the VH of SM03 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:8. The substitution, addition and / or deletion of amino acids can be in VH CDR or VL CDR. In some embodiments, amino acid substitutions, additions and / or deletions are not in CDR. In some embodiments, the variant of SM03 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of SM03 has up to 3 conservative amino acid substitutions.

[0311] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH from SM06. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL including VL CDR 1, 2 and 3 of the VL from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH including VHCDR 1, 2 and 3 of the VH from SM06. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDR 1, 2 and 3 and a VH including VH CDR 1, 2 and 3, which are respectively from the VL and VH of SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are variants of SM03. The VL of the SM06 variant can be a variant of the VL of SM06 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:9. The VH of the SM06 variant can be a variant of the VH of SM06 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:10. The amino acid substitutions, additions and / or deletions can be in the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of SM06 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of SM06 have up to 3 conservative amino acid substitutions.

[0312] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL from hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH from hLL2. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL including VL CDR 1, 2 and 3 of the VL from hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH including VHCDR 1, 2 and 3 of the VH from hLL2. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDR 1, 2 and 3 and a VH including VH CDR 1, 2 and 3, which are respectively from the VL and VH of hLL2. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are variants of hLL2. The VL of the hLL2 variant can be a variant of the VL of hLL2 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 19. The VH of the hLL2 variant can be a variant of the VH of hLL2 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 20. The amino acid substitutions, additions and / or deletions can be in the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of hLL2 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of hLL2 have up to 3 conservative amino acid substitutions.

[0313] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VL from gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VH from gemtuzumab. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have both a VL and a VH from gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL of gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VH CDRs 1, 2, and 3 of the VH of gemtuzumab. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are respectively from the VL and VH of gemtuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are variants of gemtuzumab. The VL of the gemtuzumab variant can be a variant of the VL of gemtuzumab with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 27. The VH of the gemtuzumab variant can be a variant of the VH of gemtuzumab with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 28. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of gemtuzumab have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of gemtuzumab have up to 3 conservative amino acid substitutions.

[0314] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as HuMy9-6. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have VL from HuMy9-6. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have VH from HuMy9-6. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have VL and VH from HuMy9-6 at the same time. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have VL including VLCDR 1, 2 and 3 of the VL from HuMy9-6. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have VH including VH CDR 1, 2 and 3 of the VH from HuMy9-6. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have VL including VL CDR 1, 2 and 3 and VH including VH CDR 1, 2 and 3, which are respectively from the VL and VH of HuMy9-6. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are variants of HuMy9-6. The VL of the HuMy9-6 variant can be a variant of the VL of HuMy9-6 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:35. The VH of the HuMy9-6 variant can be a variant of the VH of HuMy9-6 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:36. The amino acid substitutions, additions and / or deletions can be in the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of HuMy9-6 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of HuMy9-6 have up to 3 conservative amino acid substitutions.

[0315] In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VL from lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VH from lintuzumab. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have both a VL and a VH from lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL of lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VH CDRs 1, 2, and 3 of the VH of lintuzumab. The anti-CD33 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are respectively from the VL and VH of lintuzumab. In some embodiments, the anti-CD33 antibodies or antigen-binding fragments thereof provided herein are variants of lintuzumab. The VL of the lintuzumab variant can be a variant of the VL of lintuzumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 43. The VH of the lintuzumab variant can be a variant of the VH of lintuzumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 44. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of lintuzumab have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of lintuzumab have up to 3 conservative amino acid substitutions.

[0316] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as cLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL from cLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH from cLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from cLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL from cLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VHCDRs 1, 2, and 3 of the VH from cLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are respectively from the VL and VH of cLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are variants of cLL1. The VL of the cLL1 variant can be a variant of the VL of cLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:51. The VH of the cLL1 variant can be a variant of the VH of cLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:52. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of cLL1 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of cLL1 have up to 3 conservative amino acid substitutions.

[0317] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VH CDRs 1, 2, and 3 of the VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are respectively from the VL and VH of hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are variants of hLL1. The VL of the hLL1 variant can be a variant of the VL of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 53. The VH of the hLL1 variant can be a variant of the VH of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 54. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of hLL1 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of hLL1 have up to 3 conservative amino acid substitutions.

[0318] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDR 1, 2, and 3 of the VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VHCDR1, 2, and 3 of the VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDR 1, 2, and 3 and a VH comprising VH CDR 1, 2, and 3, which are respectively from the VL and VH of hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are variants of hLL1. The VL of the hLL1 variant can be a variant of the VL of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 53. The VH of the hLL1 variant can be a variant of the VH of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 54. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of hLL1 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of hLL1 have up to 3 conservative amino acid substitutions.

[0319] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are antibodies designated as aducanumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL from aducanumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH from aducanumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have both a VL and a VH from aducanumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL from aducanumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH comprising VH CDRs 1, 2, and 3 of the VH from aducanumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDRs 1, 2, and 3 and a VH including VH CDRs 1, 2, and 3, which are respectively from the VL and VH of aducanumab. In some embodiments, the Aβ antibodies or antigen-binding fragments thereof provided herein are variants of aducanumab. The VL of the aducanumab variant may be a variant of the VL of aducanumab having up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO:61. The VH of the aducanumab variant may be a variant of the VH of aducanumab having up to about 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO:62 or SEQ ID NO.63. The amino acid substitutions, additions, and / or deletions may be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions, and / or deletions are not in the CDR. In some embodiments, the variants of aducanumab have up to about 5 conservative amino acid substitutions. In some embodiments, the variant of aducanumab has up to 3 conservative amino acid substitutions.

[0320] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are antibodies designated as BAN2401. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL from BAN2401. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH from BAN2401. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have both a VL and a VH from BAN2401. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL from BAN2401. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH comprising VH CDRs 1, 2, and 3 of the VH from BAN2401. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are from the VL and VH of BAN2401, respectively. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are variants of BAN2401. The VL of the BAN2401 variant may be a variant of the VL of BAN2401 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 70. The VH of the BAN2401 variant may be a variant of the VH of BAN2401 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 71. The amino acid substitutions, additions and / or deletions may be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of BAN2401 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of BAN2401 have up to 3 conservative amino acid substitutions.

[0321] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are antibodies designated as gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL from gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH from gantnerumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL including VL CDRs 1, 2, and 3 of the VL from gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH including VH CDRs 1, 2, and 3 of the VH from gantnerumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDRs 1, 2, and 3 and a VH including VH CDRs 1, 2, and 3, which are respectively from the VL and VH of gantnerumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are variants of gantnerumab. The VL of the gantnerumab variant may be a variant of the VL of gantnerumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 78. The VH of the gantnerumab variant may be a variant of the VH of gantnerumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 79. The amino acid substitutions, additions and / or deletions may be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of gantnerumab have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of gantnerumab have up to 3 conservative amino acid substitutions.

[0322] In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are antibodies named cranilizumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL from cranilizumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH from cranilizumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from cranilizumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VL including VL CDRs 1, 2, and 3 of the VL of cranilizumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein have a VH including VH CDRs 1, 2, and 3 of the VH of cranilizumab. The anti-Aβ antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDRs 1, 2, and 3 and a VH including VH CDRs 1, 2, and 3, which are respectively from the VL and VH of cranilizumab. In some embodiments, the anti-Aβ antibodies or antigen-binding fragments thereof provided herein are variants of cranizumab. The VL of the cranizumab variant can be a variant of the VL of cranizumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 86. The VH of the cranizumab variant can be a variant of the VH of cranizumab having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 87. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of cranizumab have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of cranizumab have up to 3 conservative amino acid substitutions.

[0323] In some embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein is an antibody named AD38. In some embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein has a VL from AD38. In certain embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein has a VH from AD38. The anti-Aβ antibody or antigen-binding fragment thereof provided herein may have both VL and VH from AD38. In some embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein has a VL including VL CDR 1, 2 and 3 of the VL from AD38. In some embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein has a VH including VH CDR 1, 2 and 3 of the VH from AD38. The anti-Aβ antibody or antigen-binding fragment thereof provided herein may have a VL including VL CDR 1, 2 and 3 and a VH including VHCDR 1, 2 and 3, which are respectively from the VL and VH of AD38. In some embodiments, the anti-Aβ antibody or antigen-binding fragment thereof provided herein is a variant of AD38. The VL of the AD38 variant can be a variant of the VL of AD38 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:86. The VH of the AD38 variant can be a variant of the VH of AD38 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:87. The amino acid substitutions, additions and / or deletions can be in the VH CDR or VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variant of AD38 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of AD38 has up to 3 conservative amino acid substitutions.

[0324] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH from SM06. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VL including VL CDR 1, 2 and 3 of the VL from SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein have a VH including VHCDR 1, 2 and 3 of the VH from SM06. The anti-CD22 antibodies or antigen-binding fragments thereof provided herein may have a VL including VL CDR 1, 2 and 3 and a VH including VH CDR 1, 2 and 3, which are respectively from the VL and VH of SM06. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are variants of SM06. The VL of the SM06 variant can be a variant of the VL of SM06 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:9. The VH of the SM06 variant can be a variant of the VH of SM06 with up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO:10. The substitutions, additions and / or deletions of amino acids can be in VH CDR or VL CDR. In some embodiments, the substitutions, additions and / or deletions of amino acids are not in CDR. In some embodiments, the variants of SM06 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of SM06 have up to 3 conservative amino acid substitutions.

[0325] In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are antibodies designated as hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have both VL and VH from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VL comprising VL CDRs 1, 2, and 3 of the VL from hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein have a VH comprising VHCDRs 1, 2, and 3 of the VH from hLL1. The anti-CD74 antibodies or antigen-binding fragments thereof provided herein may have a VL comprising VL CDRs 1, 2, and 3 and a VH comprising VH CDRs 1, 2, and 3, which are respectively from the VL and VH of hLL1. In some embodiments, the anti-CD74 antibodies or antigen-binding fragments thereof provided herein are variants of hLL1. The VL of the hLL1 variant can be a variant of the VL of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 53. The VH of the hLL1 variant can be a variant of the VH of hLL1 having up to about 5 amino acid substitutions, additions and / or deletions in SEQ ID NO: 54. The amino acid substitutions, additions and / or deletions can be in the VH CDR or the VL CDR. In some embodiments, the amino acid substitutions, additions and / or deletions are not in the CDR. In some embodiments, the variants of hLL1 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of hLL1 have up to 3 conservative amino acid substitutions.

[0326] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments that can be used for the methods disclosed herein include VH or VL with at least one framework (FR) region. The FR region for VL can be from the Vκ10 mouse germline family. In some embodiments, the FR1, FR2, FR3, and FR4 for VL can have the amino acid sequences of SEQ ID NO:30, NO:31, NO:32, and NO:33, respectively (SM03 VL framework sequences). In some embodiments, the FR region for VH can be from the VH5 mouse germline family. In some embodiments, the FR1, FR2, FR3, and FR4 for VH can have the amino acid sequences of SEQ ID NO:34, 35, 36, and 37, respectively (SM03 VH framework sequences). In some embodiments, the FR1 region for VL can be from the VκID human germline family, the FR2 region for VL can be from the Vκ1 human germline family, the FR3 region for VL can be from the Vκ1 human germline family, and the FR4 region for VL can be from the VκJ1 human germline family. In some embodiments, the FR1, FR2, FR3 and FR4 for VL can have the amino acid sequences of SEQ ID NOs: 22, 23, 24 and 25, respectively (SM06 VL framework sequences). In some embodiments, the FR1 region for VH can be from V H 3 human germline families; the FR2 region for VH can be derived from V H 3 human germline families; the FR3 region for VH can be derived from V H 3 human germline families; and the FR4 region for VH can be from V H J5 human germline family. In some embodiments, FR1, FR2, FR3, and FR4 for VH can have the amino acid sequences of SEQ ID NOs: 26, 27, 28, and 29, respectively (SM06 VH framework sequence).

[0327] The present disclosure further contemplates additional variants and equivalents that are substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies or antibody fragments thereof described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. It will be appreciated by those skilled in the art that amino acid changes may alter post-translational processes of the antibody, such as changing the number or location of glycosylation sites, or changing membrane anchoring properties.

[0328] Variation can be the replacement, deletion or insertion of one or more nucleotides encoding antibodies or polypeptides, which causes the amino acid sequence to change compared to the native antibody or polypeptide sequence. In some embodiments, amino acid substitution is the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, such as conservative amino acid substitution. Insertion or deletion can be within the range of about 1 to 5 amino acids. In some embodiments, relative to the parent molecule, substitution, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions. In some embodiments, biologically useful and / or relevant variations in the amino acid sequence can be determined by systematically inserting, deleting or replacing in the sequence, and testing the activity of the resulting variant protein compared to the parent protein.

[0329] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments thereof that can be used in the methods provided herein are chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments thereof that can be used in the methods provided herein are humanized antibodies or antigen-binding fragments. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments thereof include VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and / or VH CDR3 from antibodies or antigen-binding fragments having the respective antigen specificity described herein. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments thereof include variants of the respective anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein. In some embodiments, the variants of anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments include substitutions, additions and / or deletions of 1 to 30 amino acids in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the variants of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include substitutions, additions and / or deletions of 1 to 25 amino acids in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the variants of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include substitutions, additions and / or deletions of 1 to 20 amino acids in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the variants of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include 1 to 15 substitutions, additions and / or deletions in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the variants of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include 1 to 10 substitutions, additions and / or deletions in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the variants of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include 1 to 5 conservative amino acid substitutions, additions and / or deletions in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, variants of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include substitutions, additions and / or deletions of 1 to 3 amino acids in the respective anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments. In some embodiments, the amino acid substitutions, additions and / or deletions are conservative amino acid substitutions.In some embodiments, conservative amino acid substitutions are in the CDRs of the antibody or antigen-binding fragment. In some embodiments, conservative amino acid substitutions are not in the CDRs of the antibody or antigen-binding fragment. In some embodiments, conservative amino acid substitutions are located in the framework regions of the antibody or antigen-binding fragment.

[0330] As known in the art, the constant region of an antibody mediates a variety of effector functions, and these effector functions may vary depending on the isotype of the antibody. For example, the C1 component of complement binds to the Fc region of an IgG or IgM antibody (binding to an antigen) to activate the complement system. Complement activation is important for the conditioning and lysis of cellular pathogens. The activation of complement also stimulates inflammatory responses and may participate in autoimmune hypersensitivity reactions. In addition, the Fc region of an antibody may bind to cells expressing Fc receptors (FcRs). There are several Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Antibodies bind to Fc receptors on the cell surface to trigger many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular toxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgA antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgD antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgE antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgG antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgM antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgG1 antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgG2 antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgG3 antibody. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein include at least one constant region of a human IgG4 antibody.

[0331] In some embodiments, at least one or more constant regions have been modified or deleted in the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein. In some embodiments, the antibody includes modification of one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and / or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes more than one human constant region. In some embodiments, the modification of the constant region includes the addition, deletion or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain has been removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer that provides some molecular flexibility that is usually conferred by the non-existent constant region. In some embodiments, the modified antibody includes a CH3 domain fused directly to the hinge region of the antibody. In some embodiments, the modified antibody comprises a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0332] In some embodiments, anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include an Fc region. In some embodiments, the Fc region is fused by a hinge. The hinge can be an IgG1 hinge, an IgG2 hinge or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3 and IgG4 are known to those of ordinary skill in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, modified antibodies (e.g., modified Fc regions) provide altered effector functions, thereby affecting the biological profile of the antibody. For example, in some embodiments, the Fc region of the antibody or antigen-binding fragment provided herein is modified to enhance its ability to cross the blood-brain barrier (BBB). For example, in some embodiments, the deletion or inactivation of the constant region (by point mutation or other methods) reduces the Fc receptor binding of the modified antibody during its circulation. In some embodiments, constant region modifications reduce the immunogenicity of the antibody. In some embodiments, constant region modifications increase the serum half-life of the antibody. In some embodiments, constant region modifications reduce the serum half-life of the antibody. In some embodiments, constant region modifications reduce or remove ADCC and / or complement dependent cytotoxicity (CDC) of antibodies. In some embodiments, specific amino acids in the human IgG1 Fc region are substituted with corresponding IgG2 or IgG4 residues to reduce the effector functions (e.g., ADCC and CDC) of the modified antibodies. In some embodiments, the antibody does not have one or more effector functions (e.g., "no effector" antibodies). In some embodiments, the antibody has no ADCC activity and / or CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody has no effector function. In some embodiments, constant region modifications increase or enhance ADCC and / or CDC of antibodies. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids to provide one or more cytotoxins, oligosaccharides, or carbohydrate attachment sites. In some embodiments, anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments include variant Fc regions that are engineered to replace specific amino acid positions compared to native Fc regions. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein comprise an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from K214R, L234A, L235E, G237A, D356E and L358M according to EU numbering.In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E and L358M according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, C226S, C229S and P238S according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, D356E and L358M according to EU numbering. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of S131C, K133R, G137E, G138S, Q196K, I199T, N203D, K214R, C226S, C229S and P238S according to EU numbering.

[0333] In some embodiments, variants may include adding amino acid residues at the amino and / or carboxyl terminal of an antibody or polypeptide. The length of the extra amino acid residues may range from one residue to one hundred or more residues. In some embodiments, variants include N-terminal methionyl residues. In some embodiments, variants include extra polypeptides / proteins (e.g., Fc regions) to produce fusion proteins. In some embodiments, variants are engineered to detect, and may include detectable markers and / or proteins (e.g., fluorescent tags or enzymes).

[0334] Therapeutic antibodies can be redesigned to promote trans-BBB transport through various mechanisms, including, for example, receptor-mediated transcytosis, adsorptive transcytosis, carrier-mediated transport, intercellular transport, and diffusion. Among them, receptor-mediated transport (RMT) has been widely used to promote the entry of antibodies. The use of RMT includes transferrin receptor, insulin receptor, low-density lipoprotein receptor (LDL receptor), CD98, TMEM50A and other endothelial cell surface receptors, which can be transported after binding. Antibody binding to transferrin, insulin receptor, CD98 and TMEM50A can cause transcytosis of the entire complex. In the case of the LDL receptor, binding to apolipoprotein can trigger the transcytosis process. Therefore, therapeutic antibodies linked to antibody fragments or apolipoproteins have been proposed and verified to enhance BBB crossing in animal models.

[0335] Therefore, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein can be designed to enhance their ability to cross the BBB. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein are fused to a second antibody or antigen binding fragment that binds to transferrin, insulin receptor, CD98 or TEME50A. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein can be fused with a second antibody or antigen binding fragment that binds to transferrin. In some embodiments, for example, anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments can be fused with a Fab fragment that binds to transferrin receptor. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided by the present invention can be fused with a second antibody or antigen binding fragment that binds to insulin receptor. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein can be fused with a second antibody or antigen binding fragment that binds CD98. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein can be fused with a second antibody or antigen binding fragment that binds TEME50A. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen binding fragments provided herein are fused with apolipoprotein.

[0336] The variant antibodies or antigen-binding fragments described herein can be produced using methods known in the art, including, but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0337] In some embodiments, variants of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments disclosed herein may retain the ability to bind to their corresponding antigen targets to a similar, identical or higher degree as the parent antibody or antigen-binding fragment. In some embodiments, the amino acid sequence of the variant may be at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical to the parent antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments include the amino acid sequences of the respective parent anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments with one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties.

[0338] In some embodiments, the variant of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibody or antigen-binding fragment comprises an amino acid sequence of a parent antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions. In some embodiments, the variant of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibody or antigen-binding fragment comprises an amino acid sequence of a parent binding antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., CD22 binds to a variant of an anti-CD22 bispecific antibody or antigen-binding fragment, CD33 binds to a variant of an anti-CD33 bispecific antibody or antigen-binding fragment, or CD74 binds to a variant of an anti-CD74 bispecific antibody or antigen-binding fragment). In certain embodiments, one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions can enhance the biological activity of the variant, thereby improving the biological activity of the functional variant compared to the parent binding portion.

[0339] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein are chemically modified naturally or by intervention. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage and / or connection to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques. The anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments may include one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0340] The anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments disclosed herein can be analyzed for their physical, chemical and / or biological properties by various methods known in the art. In some embodiments, the ability of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies to bind to their corresponding antigens, such as CD22 (e.g., human CD22), CD33 or CD74, is detected. Binding assays include, but are not limited to, surface plasmon resonance (e.g., Biacore), ELISA, and FACS. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) with its corresponding antigen is a dissociation constant determined by surface plasmon resonance (e.g., BIAcore). In addition, the solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency of the antibody can also be evaluated.

[0341] All anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein bind to their corresponding antigens with a dissociation constant (K) ranging from about 0.02 to 10 nM. D In some embodiments, the dissociation constant (K) of the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein for binding to their corresponding antigens is D ) is about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, K D is about 20 nM or less. In some embodiments, K D is about 10 nM or less. In some embodiments, K D is about 5 nM or less. In some embodiments, K D is about 2 nM or less. In some embodiments, K D is about 1.5 nM or less. In some embodiments, K D is about 1 nM or less. In some embodiments, K D is about 0.5 nM or less. In some embodiments, K D is about 0.1 nM or less. In some embodiments, K D is about 50 pM or less. In some embodiments, K D About 10 pM or less.

[0342] In some embodiments, the K of anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments binding to their corresponding antigens D In the range of 0.1-1 nM, 0.5-5 nM, 1-10 nM, 1-5 nM, 5-50 nM, 10-100 nM or 50-500 nM. In some embodiments, K D In the range of 0.1-1 nM. In some embodiments, K D In the range of 0.5-5 nM. In some embodiments, K D In the range of 1-10 nM. In some embodiments, K D In the range of 1-5 nM. In some embodiments, K D In the range of 5-50 nM. In some embodiments, K D In the range of 10-100 nM. In some embodiments, K D In the range of 50-500nM.

[0343] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to their respective antigens with an association constant (K A ) is about 0.8x10 9 M -1 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to their corresponding antigens, and their K A About 1×10 6 M -1 or higher, about 1×10 7 M -1 or higher, about 1×10 8 M -1 or higher, about 5×10 8 M -1 or higher, about 8×10 8 M -1 or higher, about 1×10 9 M -1 or higher, about 5×10 9 M -1 or higher, about 1×10 10 M -1 or higher, about 5×10 10 M -1 or higher, about 1×10 11 M -1 or higher, about 5×10 11 M -1 or higher, or about 1×10 12 M -1 Or higher. In some embodiments, K A About 1×10 7 M -1 Or higher. In some embodiments, K A About 5×10 7 M -1 Or higher. In some embodiments, K A About 1×10 8 M -1 Or higher. In some embodiments, K A About 5×10 8 M -1 Or higher. In some embodiments, K A About 8×10 8 M -1 Or higher. In some embodiments, K A About 1×10 9 M -1 Or higher. In some embodiments, KA About 5×10 9 M -1 Or higher. In some embodiments, K A About 1×10 10 M -1 or higher.

[0344] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to their corresponding antigens, and their K A In about 1×10 6 -1×10 7 M -1 , 5×10 6 -5×10 7 M -1 , 1×10 7 -1×10 8 M -1 , 5×10 7 -5×10 8 M -1 , 1×10 8 -5×10 8 M -1 , 1×10 8 -1×10 9 M -1 , 5×10 8 -1×10 9 M -1 , 5×10 8 -5×10 9 M -1 , 1×10 9 -1×10 10 M -1 , 5×10 9 -5×10 10 M -1 , 1×10 10 -1×10 11 M -1 , 5×10 10 -5×10 11 M -1 , 1×10 11 -1×10 12 M -1 or 5×10 11 -5×10 12 M -1 In some embodiments, K A In about 1×10 6 -1×10 7 M -1In some embodiments, K A In about 1×10 7 -1×10 8 M -1 In some embodiments, K A In about 1×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -5×10 9 M -1 In some embodiments, K A In about 1×10 9 -1×10 10 M -1 within the range.

[0345] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from their corresponding human antigens with a kd of 0.0685 RU s -1 or less, or 0.0137RUs -1 or less, as determined by SPR (e.g., BIAcore). In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from their corresponding human antigens with a kd of about 0.5 RU s -1 Or smaller, about 0.2RU s -1 Or smaller, about 0.1RU s -1 Or lower, about 0.08RU s -1 Or lower, about 0.06RU s -1 Or lower, about 0.05RU s -1 Or lower, about 0.04RU s -1 Or lower, about 0.03RU s -1 Or lower, about 0.02RU s -1 Or lower, about 0.01RU s -1 Or lower, about 0.008RU s -1 Or lower, about 0.005RU s -1 or lower, or about 0.001RU s -1In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from their corresponding human antigens with a kd of about 0.5 RU s-1. 1 In some embodiments, kd is about 0.2 RU s -1 In some embodiments, kd is about 0.1 RU s -1 In some embodiments, kd is about 0.08 RU s -1 In some embodiments, kd is about 0.06 RU s -1 In some embodiments, kd is about 0.05 RU s -1 In some embodiments, kd is about 0.02 RU s -1 In some embodiments, kd is about 0.01 RU s -1 In some embodiments, kd is about 0.005 RU s -1 or smaller.

[0346] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from their corresponding human antigens with a kd range of 0.001-0.5 RU s -1 、0.001-0.1RU s -1 、0.001-0.05RU s -1 、0.005-0.5RU s -1 、0.005-0.1RU s -1 , 0.01-0.5RU s -1 , 0.01-0.1RU s -1 or 0.01-0.05RU s -1 In some embodiments, kd is between 0.005-0.05 RU s -1 In some embodiments, kd is between 0.01-0.1 RU s -1 In some embodiments, kd is between 0.005-0.1 RU s -1 In some embodiments, kd is between 0.01-0.5 RU s -1 In some embodiments, kd is between 0.01-0.1 RU s -1 within the range.

[0347] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to their corresponding human antigens with a ka of 1.13×10 7 R U S -1 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to their corresponding human antigens with a ka of about 1.0×10 5 R U S -1 or higher, about 5.0×10 5 R U S -1 or higher, about 1.0×10 6 R U S -1 or higher, about 5.0×10 6 R U S -1 or higher, about 1.0×10 7 R U S -1 or higher, about 2.0×10 7 R U S -1 or higher, about 3×10 7 R U S -1 or higher, about 4.0×10 7 R U S -1 or higher, about 5.0×10 7 R U S -1 or higher, about 1.0×10 8 R U S -1 or higher, about 5.0×10 8 R U S -1 or higher, or about 1.0×10 9 R U S -1 or higher. In some embodiments, ka is about 1.0×10 6 R U S -1 or higher. In some embodiments, ka is about 5.0×10 6 R U S -1 or higher. In some embodiments, ka is about 1.0×10 7 R U S -1 or higher. In some embodiments, ka is about 2.0×10 7 R U S -1 or higher. In some embodiments, ka is about 5.0×10 7 R U S -1 or higher. In some embodiments, ka is about 1.0×10 8 R U S -1or higher. In some embodiments, ka is about 1.0×10 9 R U S -1 or higher.

[0348] In some embodiments, ka is between 1.0×10 5 -1.0×10 6 R U S -1 , 1.0×10 6 -1.0×10 7 R U S -1 5.0×10 6 -5.0×10 7 R U S -1 , 1.0×10 7 -5.0×10 7 R U S -1 , 1.0×10 7 -1.0×10 8 R U S -1 , or 1.0×10 8 -1.0×10 9 R U S -1 In some embodiments, ka is within the range of 1.0×10 6 -1.0×10 7 R U S -1 In some embodiments, ka is within the range of 5.0×10 6 -5.0×10 7 R U S -1 In some embodiments, ka is within the range of 1.0×10 7 -5.0×10 7 R U S -1 In some embodiments, ka is within the range of 1.0×10 7 -1.0×10 8 R U S -1 In some embodiments, ka is within the range of 1.0×10 8 -1.0×10 9 R U S -1 within the range.

[0349] Epitope mapping is a method for identifying binding sites, regions or epitopes on a target protein to which an antibody binds. A variety of methods for epitope mapping on a target protein are known in the art. These methods include mutagenesis, including but not limited to shotgun mutagenesis, site-directed mutagenesis and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display, and ribosome / mRNA display); methods involving protein hydrolysis and mass spectrometry; and structural determination (e.g., X-ray crystallography and NMR). In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein are characterized by assays including but not limited to N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography, and papain digestion.

[0350] The anti-CD22, anti-CD33 and / or anti-CD74 antibodies used in the methods disclosed herein can bind to linear, continuous conformational or discontinuous conformational epitopes of their corresponding antigens. Antibodies with the same target but different epitope specificities will exhibit different properties, such as affinity, induced internalization rate, induction of downstream signaling pathways to cause anti-inflammatory or immunomodulatory responses, etc. For ease of illustration, anti-CD22 antibodies SM03, SM06 and hLL2 will be used as examples. As described in Leung et al., (2015) Mabs7(1):66-76; Zhao et al., (2014) PLOS ONE 9(5):e96697; U.S. Patent No. US 9371396B2; and Chinese Patent No. ZL201210286457.4, the entire contents of which are incorporated herein by reference, SM03 and SM06 bind to domain 2 of human CD22 with high affinity, particularly to domains containing the sequence 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 (SEQ ID NO: 18) with a discontinuous conformational epitope binding affinity (Ka) of 0.82×10 9 M -1 The binding of this conformational epitope can induce rapid internalization of CD22 (e.g., 50% of surface CD22 can be internalized within 10 minutes), promote the cis-trans conversion of 2,6-sialic acid binding of CD22, and restore immune tolerance. -Orbea J et al., (2017) Molecular basis of human CD22 function and therapeutic targeting. Nat Commun. 8(1): 764, epratuzumab binds to domains 2 and 3 of CD22. From internal data, epratuzumab exhibits a much slower CD22 internalization rate than SM03 and SM06.

[0351] Thus, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments useful in the methods disclosed herein can bind to epitopes of the corresponding antigens, thereby (a) inducing internalization of surface receptors / antigens; and / or (b) inhibiting the activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation; and / or (c) reducing pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) modulating antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells; and / or (e) promoting remyelination of damaged myelin by oligodendrocytes or other neuroimmune cells.

[0352] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments useful in the methods disclosed herein can be present in an amount of about 1-10×10 9 M -1 K in the range A Binds to a linear or conformational epitope of the corresponding antigen. In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein can bind to a linear or conformational epitope of the corresponding antigen. 7 M -1 or above, about 1×10 8 M -1 or above, about 5×10 8 M -1 or above, about 1×10 9 M -1 or above, about 5×10 9 M -1 or above, about 1×10 10 M -1 or above, about 5×10 10 M -1 or above, about 1×10 11 M -1 or above, about 5×10 11 M -1 or more, or about 1×10 12 M -1 or above K AIn some embodiments, K A About 1×10 7 M -1 or more. In some embodiments, K A About 5×10 7 M -1 or more. In some embodiments, K A About 1×10 8 M -1 or more. In some embodiments, K A About 5×10 8 M -1 or more. In some embodiments, K A About 8×10 8 M -1 or more. In some embodiments, K A About 1×10 9 M -1 or more. In some embodiments, K A About 5×10 9 M -1 or more.

[0353] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments used in the methods disclosed herein bind to a linear or conformational epitope of the corresponding antigen, and their K A In about 1×10 6 -1×10 7 M -1 , 5×10 6 -5×10 7 M -1 , 1×10 7 -1×10 8 M -1 , 5×10 7 -5×10 8 M -1 , 1×10 8 -5×10 8 M -1 , 1×10 8 -1×10 9 M -1 , 5×10 8 -1×10 9 M -1 , 5×10 8 -5×10 9 M -1 , 1×10 9 -1×10 10 M -1 , 5×109 -5×10 10 M -1 , 1×10 10 -1×10 11 M -1 , 5×10 10 -5×10 11 M -1 , 1×10 11 -1×10 12 M -1 or 5×10 11 -5×10 12 M -1 In some embodiments, K A In about 1×10 6 -1×10 7 M -1 In some embodiments, K A In about 1×10 6 -1×10 7 M -1 In some embodiments, K A In about 1×10 7 -1×10 8 M -1 In some embodiments, K A In about 1×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -1×10 9 M -1 In some embodiments, K A In about 5×10 8 -5×10 9 M -1 In some embodiments, K A In about 1×10 9 -1×10 10 M -1 within the range.

[0354] In some embodiments, antibodies or antigen-binding fragments that compete with the antibodies or antigen-binding fragments provided above for binding to CD22 (e.g., human CD22), CD33 (e.g., human CD33), or CD74 (e.g., human CD74) are also provided herein. An antibody that "competes with another antibody for binding to a target" refers to an antibody that (partially or completely) inhibits the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of another antibody to a target, can be determined using known competition assays, such as Surface plasmon resonance (SPR) analysis. In some embodiments, an anti-CD22, anti-CD33 or anti-CD74 antibody or antigen-binding fragment competes with another antibody or antigen-binding fragment and inhibits the binding of the other antibody or antigen-binding fragment to the corresponding CD22 (e.g., human CD22), CD33 (e.g., human CD33) or CD74 (e.g., human CD74) by at least 50%, 60%, 70%, 80%, 90% or 100%. Competition assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.H01 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0355] In some embodiments, provided herein are bispecific antibodies or antigen binding fragments that compete with SM03, SM06 or hLL2 for binding to human CD22. In some embodiments, provided herein are bispecific antibodies or antigen binding fragments that compete with gemtuzumab, HuMy9-6, lintuzumab for binding to human CD33. In some embodiments, provided herein are bispecific antibodies or antigen binding fragments that compete with cLL1 or hLL1 for binding to human CD74. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments used in the methods disclosed herein compete for binding to their corresponding antigens, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 - hLL2 binds to native CD22 on the human Burkitt's lymphoma cell line Ramos cells; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125- Lintuzumab binds to native CD33 on the human monocytic cell line THP-1; or radiolabeled I 125 -cLL1 or I 125 -hLL1 binds to native CD74 on the human myeloid leukemia cell line K-562, EC 50 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein compete with their corresponding antigens for binding, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 -hLL2 binds to native CD22 on the human Burkitt lymphoma cell line Ramos cells; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125 - Lintuzumab binds to native CD33 on the human monocytic cell line THP-1; or radiolabeled I 125 -cLL1 or I 125 -hLL1 binds to native CD74 on the human myeloid leukemia cell line K-562, EC 50 About 10 μg / ml or less, or about 1 μg / ml. In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein compete with their corresponding antigens for binding, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 -hLL2 binds to native CD22 on the human Burkitt lymphoma cell line Ramos cells; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125 - Lintuzumab binds to native CD33 on the human monocytic cell line THP-1; or radiolabeled I 125 -cLL1 or I 125 -hLL1 binds to native CD74 on the human myeloid leukemia cell line K-562, EC 50 About 0.1 μg / ml or less, about 0.2 μg / ml or less, about 0.5 μg / ml or less, about 0.8 μg / ml or less, about 1 μg / ml or less, about 2 μg / ml or less, about 5.0 μg / ml or less, about 8 μg / ml or less, about 10 μg / ml or less, or about 50 μg / ml or less. In some embodiments, EC 50About 0.1 g / ml or less. In some embodiments, EC 50 About 0.5 μg / ml or less. In some embodiments, EC 50 About 1 μg / ml or less. In some embodiments, EC 50 About 2 μg / ml or less. In some embodiments, EC 50 About 5 μg / ml or less. In some embodiments, EC 50 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein compete with their corresponding antigens for binding, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 -hLL2 binds to native CD22 on the human Burkitt's lymphoma cell line Ramos cells; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125 - Lintuzumab binds to native CD33 on the human monocytic cell line THP-1; or radiolabeled I 125 -cLL1 or I 125 -hLL1 binds to native CD74 on the human myeloid leukemia cell line K-562, and its EC 50 In the range of about 0.1-50 μg / ml, about 0.1-10 μg / ml, about 0.1-5 μg / ml, about 0.5-10 μg / ml, about 0.5-5 μg / ml, about 1-10 μg / ml, or about 1-5 μg / ml. In some embodiments, EC 50 In the range of about 0.1-50 μg / ml. In some embodiments, EC 50 In the range of about 0.1-10 μg / ml. In some embodiments, EC 50 In the range of about 0.1-5 μg / ml. In some embodiments, EC 50 In the range of about 0.5-10 μg / ml. In some embodiments, EC 50 In the range of about 0.5-5 μg / ml. In some embodiments, EC 50 In the range of about 1-10 μg / ml. In some embodiments, EC 50 In the range of about 1-5 μg / ml.

[0356] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein compete with their corresponding antigens for binding, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 -hLL2; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125 -Lintuzumab; or radiolabeled I 125 -cLL1 or I 125 - hLL1 binds to their corresponding native antigens on microglia (e.g. HMC-3 cells) or / and oligodendrocyte cell lines (e.g. HOG or MO3.13 cells), and its EC 50 About 0.1 μg / ml or less, about 0.2 μg / ml or less, about 0.5 μg / ml or less, about 0.8 μg / ml or less, about 1 μg / ml or less, about 2 μg / ml or less, about 5.0 μg / ml or less, about 8 μg / ml or less, about 10 μg / ml or less, or about 50 μg / ml or less. In some embodiments, EC 50 About 0.1 μg / ml or less. In some embodiments, EC 50 About 0.2 μg / ml or less. In some embodiments, EC 50 About 0.5 μg / ml or less. In some embodiments, EC 50 About 1 μg / ml or less. In some embodiments, EC 50 About 2 μg / ml or less. In some embodiments, EC 50 About 5 μg / ml or less. In some embodiments, EC 50 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments used in the methods disclosed herein compete with their corresponding antigens for binding, wherein the radiolabeled I 125 -SM03,I 125 -SM06 or I 125 -hLL2; or radiolabeled I 125 - Gemtuzumab, I 125 -HuMy9-6 or I 125 -Lintuzumab; or radiolabeled I 125 -LL1 or I 125- hLL1 binds to their corresponding native antigens on microglia (e.g. HMC-3 cells) or / and oligodendrocyte cell lines (e.g. HOG or MO3.13 cells), and its EC 50 In the range of about 0.1-50 μg / ml, about 0.1-10 μg / ml, about 0.1-5 μg / ml, about 0.5-10 μg / ml, about 0.5-5 μg / ml, about 1-10 μg / ml, or about 1-5 μg / ml. In some embodiments, EC 50 In the range of about 0.1-50 μg / ml. In some embodiments, EC 50 In the range of about 0.1-10 μg / ml. In some embodiments, EC 50 In the range of about 0.1-5 μg / ml. In some embodiments, EC 50 In the range of about 0.5-10 μg / ml. In some embodiments, EC 50 In the range of about 0.5-5 μg / ml. In some embodiments, EC 50 In the range of about 1-10 μg / ml. In some embodiments, EC 50 In the range of about 1-5 μg / ml.

[0357] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein can induce internalization of about 20%, about 30%, about 40%, about 50%, about 60%, about 70% of the corresponding surface antigen (i.e., CD22, CD33 or CD74) within 10 minutes (min) after contact with cells (e.g., B cells, monocytes, macrophages, dendritic cells, oligodendrocytes or microglia). In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33 or CD74) within 10 min is about 30%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33 or CD74) within 10 min is about 40%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33 or CD74) within 10 min is about 50%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 60%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 70%. In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen-binding fragments provided herein can induce about 20% to 70%, about 30% to 70%, about 40% to 70%, about 30% to 60%, or about 40% to 60% internalization of the corresponding surface antigen (e.g., CD22, CD33, or CD74) within 10 min after contact. In some embodiments, the internalization rate of the surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 20% to 70%. In some embodiments, the internalization rate of the surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 40% to 70%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 30% to 60%. In some embodiments, the internalization rate of the corresponding surface antigen (e.g., CD22, CD33, or CD74) within 10 min is about 40% to 60%.

[0358] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments provided herein can be induced to internalize about 50% of the corresponding surface antigens (e.g., CD22, CD33 or CD74) within about 2 min, about 5 min, about 10 min, about 15 min, about 20 min, about 30 min, or about 1 hour after contact with a cell (e.g., a B cell, a monocyte, a macrophage, a dendritic cell, an oligodendrocyte or a microglia). In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33 or CD74) are internalized within 2 min. In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33 or CD74) are internalized within 5 min. In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33 or CD74) are internalized within 10 min. In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33, or CD74) are internalized within 15 min. In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33, or CD74) are internalized within 20 min. In some embodiments, 50% of the corresponding surface antigens (e.g., CD22, CD33, or CD74) are internalized within 30 min.

[0359] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments provided by the present invention that also exhibit binding specificity for Aβ protein can induce Aβ to internalize at a rate of about 0.2pg / s, about 0.5pg / s, about 0.8pg / s, about 1.0pg / s, about 2.0pg / s, about 5.0pg / s, about 8.0pg / s, about 10.0pg / s or about 10.0pg / s. In some embodiments, the rate is about 0.2pg / s. In some embodiments, the rate is about 0.5pg / s. In some embodiments, the rate is about 0.8pg / s. In some embodiments, the rate is about 1.0pg / s. In some embodiments, the rate is about 2.0pg / s. In some embodiments, the rate is about 2.0pg / s. In some embodiments, the rate is about 5.0pg / s. In some embodiments, the rate is about 1.0pg / s. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein can induce Aβ internalization at a rate of about 0.2-20pg / s, about 0.2-10pg / s, about 0.2-5pg / s, about 0.2-2pg / s, about 0.5-20pg / s, about 0.5-10pg / s, about 0.5-5pg / s, about 0.5-2pg / s, about 1-20pg / s, about 1-10pg / s, about 1-5pg / s or about 1-2pg / s. In some embodiments, the rate is about 0.2-20pg / s. In some embodiments, the rate is about 0.2-10pg / s. In some embodiments, the rate is about 0.5-10pg / s. In some embodiments, the rate is about 0.5-5pg / s. In some embodiments, the rate is about. In some embodiments, the rate is about 1-2pg / s.

[0360] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments provided herein that also exhibit binding specificity for Aβ proteins can be derivatized or linked to another functional molecule (e.g., another peptide or protein) and used in the methods disclosed herein. Therefore, in some embodiments, the antibodies and antigen-binding fragments used in the methods disclosed herein include derivatized and otherwise modified forms of the bispecific antibodies described herein, including immunoadhesion molecules. For example, antibodies and antigen-binding fragments can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or introduction of artificial amino acids / functional groups suitable for site-specific conjugation) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or antigen-binding fragment with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0361] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to generate trispecific or multispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linking agents having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linking agents can be purchased from Thermo Scientific of Waltham, Massachusetts.

[0362] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibody or antigen binding fragment is conjugated to a cytotoxic agent or moiety. In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibody or antigen binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate). In some embodiments, the cytotoxic moiety is a chemotherapeutic agent, including but not limited to methotrexate, doxorubicin / doxorubicin, phenylalanine mustard, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines, oxadiazine, chlorambucil, doxycycline ... Class (PBD) or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor, including but not limited to auristatins, maytansinoids (e.g., DM1 and DM4) and tubulysins. In some embodiments, the cytotoxic moiety is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, three-year tung protein, dianthin protein, vertical order pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, saponin inhibitor, gelonin, Mitogellin, restrictocin, phenomycin, enomycin and trichothecenes. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as kachitoxin, maytansin, trichothecenes and CC1065.

[0363] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein are conjugated to a detectable substance or molecule that allows the agent to be used for diagnosis and / or detection. Detectable substances may also include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and flavin; fluorescent materials such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), 5-dimethylamine-1-naphthalenesulfonyl chloride, and phycoerythrin; bioluminescent materials such as luciferase; radioactive materials such as 212 Bi, 14 C. 57 Co. 51 Cr, 67 Cu, 18 F. 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge, 3 H. 166 Ho, 131 I. 125 I. 123 I. 121 I. 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P. 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Such as 35 S. 47 Sc, 75 Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr. 99m Tc, 201 Ti, 133 Xe, 90 Y. 69 Yb, 175 Yb, 65Zn; positron emitting metals; and magnetic metal ions Positron emitting metals; and magnetic metal ions.

[0364] The anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments described herein may be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. In some embodiments, immobilized anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments are used for immunoassays. In some embodiments, immobilized anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments are used for purification of corresponding target antigens (e.g., human CD22, human CD33 or human CD74).

[0365] 7.2.2 Other CD22 Antibodies

[0366] Disclosed herein are methods for promoting clearance of Aβ plaques, methods for reducing neuroinflammation, methods for preventing synaptic phagocytosis and / or neuronal death, methods for treating Aβ-related diseases or disorders, and methods for treating diseases or disorders associated with neuroinflammation using a therapeutically effective amount of an anti-CD22 (e.g., human CD22), anti-CD33 (e.g., human CD33), or anti-CD74 (e.g., human CD74) bispecific antibody or antigen-binding fragment disclosed herein, or a variant or equivalent thereof. Without being bound by theory, it is understood that the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies disclosed herein can be used to achieve the above therapeutic effects because they have at least one of the following functions or activities: (1) binding to a linear or conformational epitope that promotes rapid internalization in microglia; (2) promoting clearance of extracellular Aβ captured by the anti-Aβ portion of the bispecific antibody and internalization of the captured Aβ protein in microglia; (3) inhibiting proinflammatory cytokines, such as NFκB signaling and IL-6 secretion; and (4) reducing neuroinflammation.

[0367] It should be understood that anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments can promote the rapid internalization of their corresponding antigens on microglia. The rapid internalization of these corresponding antigens will then result in the rapid internalization of bispecific antibody-bound Aβ and soluble Aβ by microglia without engaging with FcγR on the cell surface, thereby avoiding ARIA-E and ARIA-H caused by FcγR engagement. In addition, the internalization of CD22, CD33 or CD74 bispecific antibodies that also bind to Aβ can lead to the clearance of Aβ and the inhibition of inflammatory cytokines, such as NFκB signaling and IL-6 secretion, thereby leading to a reduction in neuroinflammation.

[0368] Therefore, the present invention also provides a method for selecting a bispecific antibody or antigen-binding fragment, wherein one specificity is specifically targeting CD22 (e.g., human CD22), CD33 (e.g., human CD33) or CD74 (e.g., human CD74), and the other specificity is targeting Aβ protein, for at least one of the following functions or activities, wherein the selected bispecific antibody or antigen-binding fragment can be used for a method of promoting clearance of Aβ plaques, a method of reducing neuroinflammation, a method of preventing synaptic phagocytosis and / or neuronal death, a method of treating Aβ-related diseases or disorders, and / or a method of treating a disease or disorder associated with neuroinflammation: (1) binding to an epitope that promotes antigen internalization on cells expressing the respective antigen on the cell surface, such as microglia, Ramos cells and / or B cells, optionally with an antigen internalization rate of 50% in 10 minutes; (2) promoting the internalization of Aβ in microglia, optionally with an internalization rate of 1.76 pg / s; (3) reducing neuroinflammation; (4) inhibiting proinflammatory cytokines, such as NFκB signaling and IL-6 secretion. In some embodiments, the selected antibodies or antigen binding fragments can be used in methods for promoting the clearance of Aβ plaques. In some embodiments, the selected antibodies or antigen binding fragments can be used in methods for reducing neuroinflammation. In some embodiments, the selected antibodies or antigen binding fragments can be used in methods for preventing synaptic phagocytosis and / or neuronal death. In some embodiments, the selected antibodies or antigen binding fragments can be used in methods for treating Aβ-related diseases or disorders. In some embodiments, the selected antibodies or antigen binding fragments can be used in methods for treating diseases or disorders associated with neuroinflammation.

[0369] In some embodiments, antibodies or antigen-binding fragments thereof that specifically bind to linear, continuous conformational or discontinuous conformational epitopes in different extracellular domains of respective antigens, such as CD22 (e.g., human CD22), CD33 (e.g., human CD33) or CD74 (e.g., human CD74), and induce rapid internalization as disclosed herein can be isolated by screening a recombinant combinatorial antibody library (preferably a scFv phage display library) prepared using human VL and VH cDNA prepared from mRNA derived from human lymphocytes. Methods for preparing and screening such libraries are known in the art. In addition to commercially available phage display library generation kits (e.g., GE Healthcare Life Sciences Recombinant Antibody Phage System (RAPS); and New England Biolab Ph.DTM Phage Display Library Kit, Catalog No. E8100S), examples of methods and reagents particularly suitable for generating and screening antibody display libraries can be found in, for example, U.S. Pat. No. 5,223,409 to Ladner et al.; Fuchs et al., (1991) Biotechnology 9:1369-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol. Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3579-3580; Garrard et al., (1991) Biotechnology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982.

[0370] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 bispecific antibodies or antigen-binding fragments to be used in the methods disclosed herein are further selected to have a specific concentration of 0.1×10 9 M -1 In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments are selected to bind to their corresponding antigens with a K of about 1 nM or less. DIn some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies or antigen-binding fragments to be used in the methods disclosed herein are selected to bind to their corresponding antigens at 0.1 RU s -1 or lower kd or 0.01RUs -1 The binding parameters referred to herein can be determined by any method disclosed herein or known in the art, such as by surface plasmon resonance.

[0371] In some embodiments, in order to isolate murine antibodies, recombinant antibodies or human antibodies with high affinity and low shedding rate constant for human CD22, human CD33 or CD74, the epitope imprinting method described in PCT Publication No. WO93 / 06213 is used to first select human heavy and light chain sequences with similar binding activity to the respective and corresponding antigens using a parent murine antibody or chimeric anti-CD22, anti-CD33 or anti-CD74 antibody with high affinity and low shedding rate constant for human CD22. The antibody library used in this method is preferably a scFv library prepared and screened according to the methods described in PCT Publication No. WO 92 / 01047, McCafferty et al., Nature (1990) 348:552-554 and Griffiths et al., (1993) EMBO J 12:725-734. The scFv antibody library is preferably screened using recombinant human CD22, human CD33 or human CD74 and / or recombinant fusion proteins in which the extracellular domain of each and the corresponding antigen is fused to the Fc portion of a murine or human immunoglobulin.

[0372] Once the initial human VL and VH fragments are selected, a "mix and match" experiment can be performed in which different pairings of the initially selected VL and VH fragments are screened for binding to human CD22, human CD33, or human CD74 to select preferred VL / VH pairing combinations. The affinity and / or specificity of antibody binding to a particular antigen can be improved using "directed evolution" methods, as described in Wu et al., J. Mol. Biol. Biol., 294: 151 (1999), the entire contents of which are incorporated herein by reference. In addition, to further improve the affinity for binding to human CD22, human CD33, or human CD74 and / or reduce the shedding rate constant, the VL and VH fragments of the preferred VL / VH pairs can be randomly mutated, preferably within the CDR3 regions of the VH and / or VL, in a process similar to the in vivo somatic mutation process responsible for antibody affinity maturation during the natural immune response. Such in vitro affinity maturation can be achieved by amplifying the VH and VL regions using PCR primers complementary to the VH CDR3 or VL CDR3, respectively, which are "spike" with a random mixture of four nucleotide bases at certain positions, so that the resulting PCR products encode VH and VL fragments in which random mutations have been introduced into the VH and / or VL CDR3 regions. These randomly mutated VH and VL fragments can be rescreened for binding to the extracellular domains of human CD22, human CD33 or human CD74, and / or to recombinant fusion proteins in which the extracellular domains of the respective and corresponding antigens are fused to the Fc portion of a murine or human immunoglobulin.

[0373] In addition to phage display, candidate monoclonal antibodies for screening can also be prepared using hybridoma methods well known to those skilled in the art. For example, using the hybridoma method, mice, rats, rabbits, hamsters or other appropriate host animals are immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

[0374] After immunization, lymphocytes are separated and fused with suitable myeloma cell lines using, for example, polyethylene glycol. Hybridoma cells are screened using special culture media known in the art, and unfused lymphocytes and myeloma cells cannot survive in the screening process. The hybridoma producing the monoclonal antibody for the selected antigen can be identified by a variety of methods, including but not limited to immunoprecipitation, immunoblotting and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, BLI, SPR (e.g., Biacore) and radioimmunoassay). Once hybridoma cells producing the desired specificity, affinity and / or active antibody are identified, the clone can be subcloned by limiting dilution or other techniques. Hybridoma can be cultured and propagated in vitro using standard methods, or propagated in vivo as animal ascites tumors. Monoclonal antibodies can be purified from culture media or ascites according to the standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis and dialysis.

[0375] Typically, framework residues in the framework region can be substituted with corresponding residues in the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are determined by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to determine framework residues important for antigen binding, and by sequence comparison to determine unusual framework residues at specific positions. (See Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, the entire contents of which are incorporated herein by reference).

[0376] Humanized antibodies have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domains. Therefore, humanized antibodies include one or more CDRs from non-human immunoglobulin molecules and framework regions from humans. Humanization methods of antibodies are well known in the art, including CDR transplantation (see, e.g., Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); European Patent EP 239,400; International Publication WO 91 / 09967; and U.S. Patents 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089 and 6,548,640, the entire contents of each of which are incorporated herein by reference). In such humanized antibodies, the corresponding sequence of a non-human species replaces far less than a complete human variable domain. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.Methods for producing humanized antibodies also include gluing or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al. 1994, PNAS, 91:969-973, the entire contents of each of which are incorporated herein by reference), chain replacement (see, e.g., U.S. Patent No. 5,565,332, the entire contents of which are incorporated herein by reference), and in, e.g., U.S. Patent Application Publication No. US2005 / 0042664; U.S. Patent Application Publication No. US2005 / 0048617; U.S. Patent No. 6,407,213; U.S. Patent No. 5,766,886; International Publication No. WO 9317105; Tan et al., J. Immunol., 169: 1119-25 (2002); Caldas et al., Protein Eng., 13 (5): 353-60 (2000); Morea et al., Methods, 20 (3): 267-79 (2000); Baca et al., J. Biol. Chem., 272 (16): 10678-84 (1997); Roguska et al., Protein Eng., 9 (10): 895-904 (1996); Couto et al., Cancer Res., 55 (23 Supp): 5973s-5977s (1995); Couto et al., Cancer Res., 55 (8): 1717-22 (1995); Sandhu et al., Cancer Res., 55 (23 Supp): 5973s-5977s (1995); The techniques disclosed in JS, Gene, 150(2):409-10 (1994) and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), the entire contents of each of which are incorporated herein by reference.

[0377] The selection of human variable domains (including light and heavy chains) to make humanized antibodies is to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence of rodent antibodies is screened for the entire known human variable domain sequence library. The human sequence closest to rodents is accepted as the human framework (FR) of humanized antibodies (Sims et al., J.Immunol., 151: 2296 (1993); Chothia et al., J.Mol.Biol., 196: 901 (1987), the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework that is a consensus sequence of all human antibodies from a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entireties).

[0378] Humanized antibodies can retain high affinity for target antigens and other favorable biological properties. For example, the preparation of humanized antibodies can be achieved by analyzing the process of parental sequences and various conceptual humanized products using three-dimensional models of parental sequences and humanized sequences. Three-dimensional immunoglobulin models are common and are familiar to those of ordinary skill in the art. Available computer programs illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. By examining these displays, the possible role of residues in the function of candidate immunoglobulin sequences can be analyzed, i.e., the residues that affect the ability of candidate immunoglobulins to bind target antigens can be analyzed. In this way, FR residues can be selected and combined from receptor sequences and input sequences to obtain desired antibody properties, such as increasing affinity for target antigens. In general, CDR residues are directly involved in influencing antigen binding to the greatest extent.

[0379] After screening and isolating the anti-CD22, anti-CD33 or anti-CD74 antibodies disclosed herein from the recombinant immunoglobulin display library, the nucleic acid encoding the selected antibody can be recovered from the display package (e.g., from the phage genome) and subcloned into other expression vectors by standard recombinant DNA techniques. If necessary, the nucleic acid can also be further processed to produce other antibody forms disclosed herein or known in the art (e.g., connected to nucleic acids encoding additional immunoglobulin domains such as additional constant regions). Methods for expressing recombinant human antibodies isolated by combinatorial library screening, including cloning the DNA encoding the antibody into a recombinant expression vector and introducing it into a mammalian host cell, are well known in the art.

[0380] In some embodiments, the anti-CD22, anti-CD33, or anti-CD74 antibodies or antigen-binding fragments are selected to (1) bind to a linear or conformational epitope that promotes rapid internalization in microglia; (2) promote clearance of extracellular Aβ captured by the anti-Aβ portion of the bispecific antibody and internalization of the captured Aβ protein in microglia, preferably without engaging FcγR on the cell surface; (3) inhibit proinflammatory cytokines, such as NFκB signaling and IL-6 secretion; and (4) reduce microglial neuroinflammation, or any combination thereof.

[0381] In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their function of promoting internalization of their respective and corresponding antigens. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their effect of promoting the internalization of Aβ in microglia, preferably without binding to FcγR on the cell surface. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their function of reducing neuroinflammation. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their function of inhibiting NFκB signal transduction in microglia. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their function of inhibiting microglia secretion of IL-6. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen binding fragments are selected because of their function of inhibiting neuroinflammation. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments are selected because they inhibit synaptic phagocytosis. In some embodiments, anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments are selected because they inhibit neuronal death.

[0382] The anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments described herein can be detected by, for example, standard ELISA for binding to respective and corresponding antigens, i.e., human CD22, human CD33 or human CD74. Briefly, a microplate is coated with respective and corresponding purified antigens, i.e., CD22, CD33 or CD74, and then blocked with bovine serum albumin. Diluted antibodies (e.g., plasma dilutions of antigen-immunized mice) are added to each well and incubated. After washing the plate, incubate with a secondary antibody reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies, goat-anti-human IgG Fc-specific polyclonal reagent). After washing, develop and analyze with a spectrophotometer. The serum of the immunized mice can be further screened by flow cytometry for binding to cell lines expressing human CD22, human CD33 or CD74, but not to control cell lines that do not express respective and corresponding antigens. Briefly, the binding of anti-CD22, anti-CD33, or anti-CD74 antibodies can be assessed by incubating recombinant CHO cells expressing the respective antigens with the corresponding anti-CD22, anti-CD33, or anti-CD74 antibodies. After washing the cells, binding is detected with anti-human IgG antibodies. Flow cytometric analysis can be performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The mouse with the highest titer can be used for fusion.

[0383] ELISA assays as described above can be used to screen antibodies, and thereby screen hybridomas that produce antibodies that exhibit positive reactivity with each and the corresponding immunogens of the anti-CD22, anti-CD33 or anti-CD75 antibodies disclosed herein. Hybridomas that produce antibodies that bind with high affinity to CD22, CD33 or CD74 can be subcloned and further characterized. A clone that retains the parental cell reactivity (by ELISA) can then be selected from each hybridoma, which can then be selected for cell bank preparation and antibody purification.

[0384] To purify anti-CD22, anti-CD33 or anti-CD74 antibodies, selected hybridomas can be cultured to purify monoclonal antibodies. The supernatant can be filtered and concentrated before affinity chromatography. The eluted IgG can be tested by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged and the concentration determined. The monoclonal antibody can be aliquoted and stored.

[0385] A variety of methods and assays are known in the art to determine the structural and functional properties of antibodies and antigen-binding fragments. For example, to determine whether a selected anti-CD22, anti-CD33, or anti-CD74 monoclonal antibody binds to a unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of the biotinylated MAb can be detected using a streptavidin-labeled probe. Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using ELISA plates coated with the respective and corresponding CD22, CD33, or CD74 antigens.

[0386] To test the binding of monoclonal antibodies to live cells expressing CD22, CD33 or CD74, flow cytometry can be used. Briefly, cell lines expressing the respective and corresponding antigens, i.e., CD22, CD33 or CD74 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as the primary antibody staining. The samples can be analyzed by a FACScan instrument, using light and side scatter properties to gate individual cells and determine the binding of the labeled antibody. Another optional detection can be used, which is performed using fluorescence microscopy (as a supplement or alternative to flow cytometry). The cells can be stained exactly as described above and then examined using a fluorescence microscope. This method allows the visualization of individual cells, but the sensitivity is reduced due to the density of the antigen.

[0387] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-CD22, anti-CD33, or anti-CD74 antibodies include standard assays known in the art, such as biolayer interferometry (BLI) using, for example, the Gator system (Probe Life) or the Octet-96 system (Sartorius AG), or using BIACORE TM BIACORE 2000SPR instrument (Biacore AB, Uppsala, Sweden) TM Surface plasmon resonance (SPR) analysis.

[0388] Assays for testing or screening the above functional properties of anti-CD22, anti-CD33 or anti-CD74 antibodies or antigen-binding fragments are well known in the art. Any method disclosed herein or known in the art can be used for the screening methods disclosed herein. For example, functional assays that can be used for the screening methods disclosed herein include luciferase assays for measuring NFκB signaling, ELISA assays for measuring IL-6 release, ELISA or Western Blot assays for measuring synaptic phagocytosis, and MTT assays for measuring neuronal death, all of which are disclosed in the experimental section below.

[0389] In some embodiments, the present invention also provides anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies and antigen-binding fragments identified or produced using the methods described herein, and their therapeutic uses.

[0390] 7.3 Generation method

[0391] Anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies and antigen-binding fragments thereof that can be used in the methods disclosed herein include, but are not limited to, monoclonal antibodies, chimeric antibodies, human antibodies and humanized antibodies, which can be prepared by any method disclosed herein or otherwise known in the art.

[0392] Methods for producing antibodies are well known in the art. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Manual, 1996). to ry Press, 2nd ed. 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563, 681 (Elsevier, NY, 1981), the entire contents of each of which are incorporated herein by reference.

[0393] In some embodiments, the antibodies or antigen-binding fragments useful in the methods provided herein are recombinant, i.e., prepared, expressed, produced, or isolated by recombinant means. In some embodiments, the antibodies or antigen-binding fragments disclosed herein can be prepared by, for example, introducing a recombinant expression vector into a host cell, a recombinant, a combinatorial human antibody library, an antibody isolated from an animal (e.g., a mouse) of a transgenic human immunoglobulin gene (see, e.g., Taylor, LD et al., (1992) Nucl. Acid Res. 20: 6287-95) or an antibody prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences.

[0394] In some embodiments, antibodies and antigen-binding fragments can be prepared by recombinantly expressing immunoglobulin light and heavy chain genes in host cells. In order to express antibody recombinants, one or more recombinant expression vectors are introduced into host cells, and these expression vectors carry DNA fragments of immunoglobulin light and heavy chains encoding antibodies, so that light and heavy chains are expressed in host cells, preferably, secreted into the culture medium of the host cells, and antibodies can be recovered from the culture medium. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, these genes are incorporated into recombinant expression vectors, and the vectors are introduced into host cells, such as Sambrook, Fritsch and Maniais (eds), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and U.S. Patent No. 4,816,397.

[0395] To express recombinant antibodies or antigen fragments, first obtain DNA fragments encoding light and heavy chain variable regions. These DNAs can be obtained by using polymerase chain reaction (PCR) to amplify and modify hybridomas of mouse antibody light and heavy chain variable sequences, or by oligosynthesis based on the designed amino acid sequences encoding light and heavy chain variable sequences using standard methods known to those skilled in the art. The encoded DNA sequence can be further optimized to facilitate mammalian expression of the resulting antibody.

[0396] Once the VH and VL fragments of the murine antibody are obtained, these sequences can be mutated to encode framework-patched versions, the methods of which have been described in Chinese Patent Nos. 01144894.6 and 031 23054.7 and U.S. Patent Nos. 7,321,026 B2 and 7,338,659 B2, all of which are incorporated herein by reference in their entirety.

[0397] Once DNA fragments encoding the VH and VL fragments of anti-CD22, anti-CD33 or anti-CD74 antibodies are obtained (e.g., by amplification and mutagenesis of original mouse VH and VL genes, as described above), these DNA fragments can be further manipulated by standard DNA recombinant techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein (e.g., an antibody constant region or a flexible linker). As used in this context, the term "operatively linked" is intended to mean that two DNA fragments are joined together so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0398] In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 bispecific antibodies are operably linked to antibodies or antibody fragments specific for the Aβ protein. Similarly, DNA fragments encoding the Aβ antibody VH and VL fragments are obtained (as described above, by amplifying and mutagenizing the original mouse VH and VL genes), which can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an anti-CD22, anti-CD33 or anti-CD74 antibody, via a flexible linker.

[0399] The isolated DNA encoding the VH region is converted into a full-length heavy chain gene by operably linking the VH encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). In some embodiments, the anti-CD22, anti-CD33 or anti-CD74 antibody is operably linked to the Aβ antibody, preferably in the form of scFv, connected to the C-terminus of the anti-CD22, anti-CD32 or anti-CD74 antibody CH3 domain through a flexible linker. The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, Ig4, IgA, IgE, IgM or IgD constant region, but most preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the VH encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region. The scFv encoding DNA can be further operably linked to the CH1 constant region of the Fab fragment to produce a bispecific Fab.

[0400] By operably connecting VL encoding DNA to another DNA molecule encoding the constant region CL of the light chain, the DNA encoding the VL region separated is converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat, EA, et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and the DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but the kappa constant region is most preferred.

[0401] To prepare an scFv gene, the VH and VL encoding DNA fragments are operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3) so that the VH and VL sequences can be expressed as a continuous single-chain protein in which the VL and VH regions are joined by a flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0402] In order to express antibodies or antigen-binding fragments that can be used in the methods disclosed herein, the DNA encoding partial or full-length light and heavy chains obtained as described above can be inserted into an expression vector so that the gene is operably linked to the transcription and translation control sequences. In this context, the term "operably linked" is intended to refer to connecting the antibody gene to the vector so that the transcription and translation control sequences in the vector play their intended functions of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequence are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, more typically, the two genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., connection of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end connection in the absence of restriction sites). In some embodiments, before inserting the light or heavy chain sequence of the anti-CD22, anti-CD33 or anti-CD74 antibody, the expression vector already carries the antibody constant region sequence. In some embodiments, the expression vector carrying the antibody constant region sequence comprises an scFv sequence derived from an anti-Aβ antibody operably linked to the CH3 domain sequence. For example, one method of converting the VH and VL sequences of anti-CD22, anti-CD33 or anti-CD74 antibodies into full-length antibody genes is to insert them into expression vectors encoding the heavy chain constant region and the light chain constant region, respectively, so that the VH segment is operably connected to the CH segment in the vector, and the VL segment is operably connected to the CL segment in the vector. Using standard molecular biological methods, the DNA sequence encoding the Aβ antibody scFv can be operably connected to the CH3 fragment sequence of the above-mentioned expression vector to produce a bispecific antibody expression vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0403] In addition to the antibody chain genes, the recombinant expression vectors provided herein may also carry regulatory sequences for controlling the expression of the antibody chain genes in host cells. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. For example, such regulatory sequences are described in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). As will be appreciated by those skilled in the art, the design of the expression vector, including the selection of regulatory sequences, depends on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Preferred regulatory sequences for mam...

Claims

1. A method for promoting clearance of β-amyloid (Aβ) plaques in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody or the antigen-binding fragment thereof specifically binds to an internalized antigen expressed on the surface of a neuroimmune system cell at one end and specifically binds to a toxic form of Aβ at the other end.

2. The method of claim 1, wherein the bispecific antibody or antigen-binding fragment binds to the internalized antigen expressed on the surface of a neuroimmune system cell to (a) induce internalization of surface receptors / antigens; and / or (b) inhibit activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation, and / or (c) reduce pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulate antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells.

3. The method of claims 1 and 2, wherein the bispecific antibody or antigen-binding fragment binds to the following toxic forms of Aβ: (a) monomeric form; (b) oligomeric form; (c) protofibrils; (d) protofibrils; or (e) pyroglutamate-modified forms.

4. A method for reducing neuroinflammation in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of the bispecific antibody or antigen-binding fragment of claim 1, wherein the bispecific antibody or antigen-binding fragment binds to an internalized antigen expressed on the surface of a neuroimmune system cell to (a) induce internalization of surface receptors / antigens; and / or (b) inhibit activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation, and / or (c) reduce pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulate antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells.

5. The method according to claims 1, 2 and 4, wherein the internalizing antigen is CD22, CD33 or CD74, and the cells in the neuroimmune system are microglia, oligodendrocytes or astrocytes.

6. The method of claims 1 to 5, wherein the subject suffers from clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disease caused by pathogenic Aβ peptides in blood vessels.

7. A method for treating an Aβ-related disease or disorder in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to CD22, CD33 or CD74 at one end and binds to Aβ at the other end, wherein the bispecific antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation, and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells.

8. A method for treating a neuroinflammation-related disease or disorder in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds to CD22, CD33 or CD74 at one end and binds to Aβ at the other end, wherein the antibody or antigen-binding fragment (a) induces internalization of surface receptors / antigens; and / or (b) inhibits activation of microglia, astrocytes, oligodendrocytes, pericytes, brain endothelial cells, or other neuroimmune cells that promote neuroinflammation, and / or (c) reduces pathogenic synaptic pruning of microglia, astrocytes, or other neuroimmune cells; and / or (d) regulates antigen presentation to infiltrating T cells by microglia, astrocytes, endothelial cells, or other neuroimmune cells.

9. The method of claim 7 or 8, wherein the Aβ-related or neuroinflammation-related disease or disorder is clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disease caused by pathogenic Aβ peptides in blood vessels.

10. The method of claim 9, wherein the A[beta]-related disease or disorder is Alzheimer's disease.

11. The method according to any one of claims 1 to 10, wherein the bispecific antibody or antigen-binding fragment is an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

12. The method of claim 11, wherein the antibody is an IgG1 antibody.

13. The method according to any one of claims 1 to 10, wherein the bispecific antibody or antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single domain antibody (sdAb) and heavy chain antibody (HCAb).

14. The method according to any one of claims 1 to 13, wherein the bispecific antibody or antigen-binding fragment specific for CD22 comprises any one of: a. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively, or b. a light chain variable region (VL), which includes VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; and a heavy chain variable region (VH), which includes VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

15. The method of claim 14, wherein the VL and VH of the anti-CD22 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

16. The method of claim 15, wherein the anti-CD22 binding portion of the bispecific antibody or antibody fragment is SM03.

17. The method of claim 14, wherein the VL and VH of the anti-CD22 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

18. The method of claim 17, wherein the anti-CD22 binding portion of the bispecific antibody or antibody fragment is SM06.

19. The method of claim 14, wherein the VL and VH of the anti-CD22 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID No: 17 and SEQ ID NO: 18, respectively.

20. The method of claim 19, wherein the anti-CD22 binding portion of the bispecific antibody or antibody fragment is LL2.

21. The method of claim 14, wherein the VL and VH of the anti-CD22 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID No: 19 and SEQ ID NO: 20, respectively.

22. The method of claim 19, wherein the anti-CD22 binding portion of the bispecific antibody or antibody fragment is hLL2.

23. The method according to any one of claims 1 to 13, wherein the bispecific antibody or antigen-binding fragment specific for CD33 comprises any one of: a. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively, or b. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, respectively, or c. a light chain variable region (VL), which includes VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively; and a heavy chain variable region (VH), which includes VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, respectively.

24. The method of claim 23, wherein the VL and VH of the anti-CD33 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

25. The method of claim 24, wherein the anti-CD33 binding portion of the bispecific antibody or antibody fragment is gemtuzumab.

26. The method of claim 23, wherein the VL and VH of the anti-CD33 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

27. The method of claim 26, wherein the anti-CD33 binding portion of the bispecific antibody or antibody fragment is HuMy9-6.

28. The method of claim 23, wherein the VL and VH of the anti-CD33 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 44, respectively.

29. The method of claim 28, wherein the anti-CD33 binding portion of the bispecific antibody or antibody fragment is lintuzumab.

30. The method of any one of claims 1 to 13, wherein the bispecific antibody or antigen-binding fragment having specificity for CD74 comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; and A heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having the amino acid sequences of SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, respectively.

31. The method of claim 30, wherein the VL and VH of the anti-CD74 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 52, respectively.

32. The method of claim 31, wherein the anti-CD74 binding portion of the bispecific antibody or antibody fragment is cLL1.

33. The method of claim 30, wherein the VL and VH of the anti-CD74 portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID No: 53 and SEQ ID NO: 54, respectively.

34. The method of claim 33, wherein the anti-CD74 binding portion of the bispecific antibody or antibody fragment is hLL1.

35. The method according to any one of claims 1 to 13, wherein the bispecific antibody or antigen-binding fragment specific for Aβ protein comprises any one of the following: a. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, respectively, or b. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively, or c. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77, respectively, or d. a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, respectively; and a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, respectively, or e. a light chain variable region (VL), comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 88, SEQ ID NO: 89 and SEQ ID NO: 90, respectively; and a heavy chain variable region (VH), comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 91, SEQ ID NO: 92 and SEQ ID NO: 93, respectively.

36. The method of claim 35, wherein the VL and VH of the anti-Aβ portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 61 and SEQ ID NO: 62 or SEQ ID NO: 63, respectively.

37. The method of claim 36, wherein the anti-Aβ binding portion of the bispecific antibody or antibody fragment is aducanumab.

38. The method of claim 35, wherein the VL and VH of the anti-Aβ portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 71, respectively.

39. The method of claim 38, wherein the anti-Aβ binding portion of the bispecific antibody or antibody fragment is BAN2401.

40. The method of claim 35, wherein the VL and VH of the anti-Aβ portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 78 and SEQ ID NO: 79, respectively.

41. The method of claim 40, wherein the anti-Aβ binding portion of the bispecific antibody or antibody fragment is gantnerumab.

42. The method of claim 35, wherein the VL and VH of the anti-Aβ portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 86 and SEQ ID NO: 87, respectively.

43. The method of claim 42, wherein the anti-Aβ binding portion of the bispecific antibody or antibody fragment is cranizumab.

44. The method of claim 35, wherein the VL and VH of the anti-Aβ portion of the bispecific antibody or antibody fragment have the amino acid sequences of SEQ ID NO: 94 and SEQ ID NO: 95, respectively.

45. The method of claim 44, wherein the anti-Aβ binding portion of the bispecific antibody or antibody fragment is AD38.

46. ​​The method of any one of claims 1 to 45, wherein the bispecific antibody or antigen-binding fragment is administered intravenously, intramuscularly, subcutaneously, intracranialy, intrathecally, intraventricularly, intraperitoneally, intranasally, parenterally, topically, or intradermally.

47. The method of claim 46, wherein the bispecific antibody or antigen-binding fragment is administered intravenously or subcutaneously.

48. The method of any one of claims 1 to 47, wherein the bispecific antibody or antigen-binding fragment is administered in a therapeutically effective amount in the range of 1-50 mg / kg body weight of the subject.

49. The method of claim 47, wherein the therapeutically effective amount is about 1, about 2, about 3, about 5, about 10, about 15, or about 30 mg / kg body weight of the subject.

50. The method of any one of claims 1 to 47, wherein the bispecific antibody or antigen-binding fragment is administered in a therapeutically effective amount of 300-1200 mg per dose.

51. The method of any one of claims 1 to 50, wherein the bispecific antibody or antigen-binding fragment is administered once every two weeks or once a month.

52. The method of any one of claims 1 to 51, wherein the bispecific antibody or antigen-binding fragment is administered in multiple doses.

53. The method of claim 52, wherein the bispecific antibody or antigen-binding fragment is administered in multiple doses over a period of at least three months, at least six months, or at least one year.

54. The method of any one of claims 1 to 53, wherein the bispecific antibody or antigen-binding fragment is administered in conjunction with a second therapeutic agent.

55. The method of claim 54, wherein the second therapeutic agent is a Tau aggregation inhibitor, a Tau protein modulator, a cholinesterase inhibitor, an acetylcholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, a β-secretase inhibitor, or an insulin sensitizer.

56. The method of any one of claims 1 to 55, wherein the subject is a human subject.

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