Methods for Treating Neurological Disorders

Abstract

Provided herein are methods of promoting the removal of beta-amyloid (Aβ) plaques, reducing neuroinflammation, and treating neurological disorders (e.g., Alzheimer's disease) using certain anti-CD22 antibodies or antigen-binding fragments.Exemplary antibodies, their characteristics, and methods of screening for additional therapeutic antibodies are also described herein.The present invention is based on the surprising discovery by the inventors that toxic oligomeric Aβ1-42 binds to CD22 expressed on human microglia, and that certain anti-CD22 antibodies can promote the removal of Aβ plaques by inducing the internalization of CD22.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 221,261, filed July 13, 2021, which is incorporated by reference herein in its entirety.

[0002] 1. Field The present invention relates to molecular biology and neurobiology, specifically the identification and use of antibodies in the treatment of various neurological disorders associated with amyloid-β and / or neuroinflammation, such as Alzheimer's disease, amyloidosis and β-amyloid pathology. [Background technology]

[0003] 2. Background Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ)-containing extracellular plaques. The causes leading to this condition are generally unknown. It has been shown that removal of Aβ plaques can provide clinical benefits in AD patients. However, only limited therapeutic options are available for Aβ plaque removal, which is commonly associated with vascular side effects, such as ARIA-E and ARIA-H. Thus, there is a great need for novel methods to more efficiently remove Aβ plaques and therapeutic options for AD with fewer side effects. The methods provided in the present disclosure meet this need and provide related advantages. Summary of the Invention [Means for solving the problem]

[0004] 3. Abstract Provided herein is a method of promoting removal of beta-amyloid (Aβ) plaques in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22.

[0005] Also provided herein is a method of reducing neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22.

[0006] In some embodiments of the methods provided herein, the subject has clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia, cognitive impairment, memory loss, or a vascular disorder caused by pathogenic Aβ peptides in the blood vessels.

[0007] Also provided herein is a method of 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 specifically binds CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22.

[0008] Also provided herein is a method of treating a disease or disorder associated with neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22.

[0009] In some embodiments, the Aβ-related or neuroinflammatory-related disease or disorder is clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disorder caused by pathogenic Aβ peptide in blood vessels. In some embodiments, the Aβ-related disease or disorder is Alzheimer's disease.

[0010] In some embodiments, the antibody or antigen-binding fragment used in the methods described herein is a monoclonal antibody or antigen-binding fragment.

[0011] In some embodiments, the antibody or antigen-binding fragment used in the methods disclosed herein is an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody.

[0012] In some embodiments, the 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).

[0013] In some embodiments, the antibody or antigen-binding fragment used in the methods disclosed herein is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

[0014] In some embodiments, the antibody or antigen-binding fragment used in the methods disclosed herein specifically binds to CLLNFSCYGYPIQ (SEQ ID NO: 11) and VFTRSELKFSPQWSHHGKIVTC (SEQ ID NO: 12) of human CD22.

[0015] In some embodiments, the antibody or antigen-binding fragment used in the methods disclosed herein comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2 and a VL CDR3 having the 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 a VH CDR1, a VH CDR2 and a VH CDR3 having the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0016] In some embodiments, the 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.

[0017] In some embodiments, the antibody or antigen-binding fragment used in the methods disclosed herein is a human 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.

[0018] In some embodiments of the methods disclosed herein, the antibody or antigen-binding fragment is administered intravenously, intramuscularly, subcutaneously, intracranially, intrathecally, intraventricularly, intraperitoneally, intranasally, parenterally, topically, or intradermally. In some embodiments, the antibody or antigen-binding fragment is administered intravenously. In some embodiments, the antibody or antigen-binding fragment is administered subcutaneously.

[0019] In some embodiments of the methods disclosed herein, the antibody or antigen-binding fragment is administered in a therapeutically effective amount in the range of 1-50 mg / kg of the subject's body 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 of the subject's body weight. In some embodiments, the antibody or antigen-binding fragment is administered in a therapeutically effective amount of 300-1,200 mg per dose. In some embodiments, the antibody or antigen-binding fragment is administered biweekly or monthly. In some embodiments, the antibody or antigen-binding fragment is administered in multiple doses. In some embodiments, the 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.

[0020] In some embodiments of the methods disclosed herein, the antibody or antigen-binding fragment is administered in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is an anti-beta-amyloid antibody, an anti-CD33 antibody, a tau aggregation inhibitor, a tau protein modulator, a cholinesterase inhibitor, an acetylcholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, a beta-secretase inhibitor, or an insulin sensitizer.

[0021] In some embodiments, the second therapeutic agent is an anti-beta-amyloid antibody selected from the group consisting of aducanumab, donanemab, gantenerumab, lecanemab, bapineuzumab, and solanezumab.

[0022] In some embodiments, the second therapeutic agent is an anti-CD33 antibody selected from the group consisting of AL003, gemtuzumab, lintuzumab, ozogamicin, vadastuximab talirine, and BI836858.

[0023] In some embodiments of the methods disclosed herein, the subject is a human subject.

[0024] Also provided herein is a method of inducing internalization of Aβ by microglial cells, comprising contacting the microglial cells with an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22. 4. Brief description of the drawings [Brief description of the drawings]

[0025] [Figure 1] Figure 1 provides Octet in vitro binding data showing Aβ binding to recombinant CD22 protein. As shown, Aβ bound to CD22 with a KD of 6×10−8 M.

[0026] [Diagram 2] 2 provides flow cytometry data showing Aβ binding to CD22-expressing cells. As shown, overexpression of human CD22 in HEK293 cells increased the binding of FITC-Aβ onto the surface of HEK293 cells.

[0027] [Diagram 3] Figure 3 provides immunocytochemical PLA staining of HMC-3 cells after Aβ treatment. As shown, the positive signal (arrow) indicates the physical interaction between Aβ and CD22 on the cell surface of HMC-3 cells.

[0028] [Figure 4] Figure 4 provides immunofluorescence staining of CD22 in HMC3 cells. As shown, treatment with anti-CD22 antibody SM03 induced rapid internalization of CD22.

[0029] [Diagram 5] Figure 5 provides confocal data showing the internalization of anti-CD22 antibodies. As shown, FITC-SM03 was internalized within 30 minutes after being added to HMC3 cells.

[0030] [Figure 6] 6 provides flow cytometry data on the rate of CD22 internalization induced by various anti-CD22 antibodies on HMC-3 cells. As shown, SM03 and SM06 induced the highest rate of internalization.

[0031] [Figure 7] 7 provides flow cytometry data showing the internalization of Aβ. As shown, treatment with SM03 and SM06 each enhanced the phagocytosis of FITC-Aβ.

[0032] [Figure 8] 8 provides data from a luciferase assay measuring NFκB signaling. As shown, treatment with anti-CD22 antibody SM03 Fab reduced NFκB signaling in HMC-3 cells.

[0033] [Figure 9] Figure 9 provides data from an ELISA measuring IL-6 secretion. As shown, treatment with anti-CD22 antibody SM03 Fab reduced IL-6 secretion by HMC-cells.

[0034] [Figure 10] Figure 10 provides immunocytochemical staining of 2,6-sialic acid trans-binding on HMC-3 cells. As shown, SM03 and SM06 promoted trans-binding of 2,6-sialic acid probe in HMC-3 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] 5. Detailed Description Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β (Aβ)-containing extracellular plaques. Effective treatment options are still greatly needed. Disclosed herein is the inventors' surprising discovery that toxic oligomeric Aβ1-42 binds to CD22 expressed on human microglia, and that certain anti-CD22 antibodies can promote the removal of Aβ plaques by inducing CD22 internalization. In addition, it has been found that the anti-CD22 antibodies disclosed herein can also suppress autoimmunity in the central nervous system (CNS), providing additional therapeutic benefits. Thus, in some embodiments, provided herein are methods for reducing Aβ accumulation and neuroinflammation via CD22-mediated mechanisms, as well as methods for treating related diseases or disorders (e.g., AD).

[0036] Dementia is estimated to increase from 50 million in 2010 to 113 million by 2050 worldwide. AD is the leading cause of dementia and is often combined with other neurodegenerative and cerebrospinal pathologies. Neuropathological features begin 15-20 years before overt dementia symptoms. Individuals with AD progress from their normal baseline cognitive abilities, through subtle changes in the preclinical stage, to overt symptoms of brain dysfunction called prodromal AD, and finally to AD dementia, where agnosia ultimately impairs the ability to perform activities of daily living (ADLs) previously performed independently. The preclinical stage of AD is often considered the stage that occurs before a clinical diagnosis of cognitive impairment is considered. AD dementia is a disease of synaptic dysfunction that encompasses changes at the molecular, cellular and connectome levels. Clinical manifestations in AD patients include dementia with amnesic or non-amnesic symptoms. This is often accompanied by language, visuospatial processing and executive dysfunction (Scharre, Practical Neurology (2019);Dubois et al., Alzheimers Dement (2016), 12(3): 292-323). The neuropathological hallmarks of AD are characterized by excessive neuroinflammation, accumulation of extracellular Aβ-containing plaques and hyperphosphorylated tau protein. Aβ plaques are widespread in the cortical and hippocampal regions and cause the destruction of neuronal networks through the induction of neuronal loss and the promotion of microglia-mediated neuroinflammation.

[0037] Aβ is induced by amyloid precursor protein, a transmembrane protein enriched in neuronal surface membranes. Physiologically, it is cleaved by α-secretase and later by γ-secretase to form APPsα. It functions in regulating synaptic strength through modulating calcium flux and potassium channels. In the amyloidogenic pathway, Aβ is cleaved by β-secretase and subsequently by γ-secretase to form Aβ1-42. Aβ1-42 misfolds in 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 microglial cells through pattern recognition receptors, namely Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs), and AIM2-like receptors (ALRs), to induce neuroinflammation. Aβ1-42 induces pro-inflammatory cytokines, namely IL-1β, IL-8, and TNFα, released by microglia. It also activates the NLRP3 inflammasome cascade to secrete the ASC speck protein (apoptosis-associated speck-like protein containing a caspase-1 recruitment domain) for nucleation of further aggregates of Aβ1-42.

[0038] Clearance of toxic oligomeric Aβ has been proposed to confer clinical benefit in AD patients. Of the 17 disease-modifying therapeutics in Phase III as of 2021, 29% of them target Aβ as their mechanism of action (MOA). Anti-Aβ antibodies, including aducanumab, donanemab, gantenerumab, lecanemab, bapineuzumab, and solanezumab, target various toxic forms of Aβ via Fc receptor-mediated phagocytosis and non-Fc receptor-mediated clearance of Aβ.

[0039] Aducanumab is an IgG1 mAb that selectively targets soluble oligomers and insoluble fibrils of Aβ. Aducanumab binds 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 their clearance by Fc gamma receptor-mediated phagocytosis, restoring calcium homeostasis of neuronal networks in AD patients. Aducanumab was approved by the FDA in June 2021 for the treatment of AD, as clinical studies consistently demonstrated amyloid plaque reduction.

[0040] In addition to AD, other diseases or disorders associated with abnormal accumulation of Aβ include, for example, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia, cognitive impairment, memory loss, or vascular disorders caused by pathogenic Aβ peptides in the blood vessels.

[0041] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting.

[0042] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the same meaning as commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include plural, and plural terms shall include singular.In general, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are well known and commonly used in the art.

[0043] The term "a" or "an" entity refers to one or more of that entity, for example, "a vector" is understood to refer to one or more vectors.

[0044] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B", it is intended to include "A and B", "A or B", "A" (single) and "B" (single). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C", it is intended to encompass 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 (single); B (single); and C (single).

[0045] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be construed as specifically disclosing all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range, e.g., 1-6, should be construed as specifically disclosing subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0046] Exemplary genes and polypeptides are described herein with reference to GenBank numbers, GI numbers and / or SEQ ID NOs. It is understood that those skilled in the art can easily identify homologous sequences by reference to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ). 5.1 Aβ Clearance and Neuroinflammation Reduction

[0047] CD22 is a B cell-restricted antigen belonging to the immunoglobulin (Ig) superfamily. CD22 is a type I transmembrane sialoglycoprotein, also known as sialic acid-binding immunoglobulin-type lectin (Siglec). Human CD22 specifically binds to the following structures: N-acetylneuraminic acid alpha(2-6)galactose (NeuAc-alpha(2-6)Gal), which is known to be expressed on hematopoietic cells, liver cells, lung epithelial cells, splenic cords and pulp, ileal stoma, cardiac stroma, blood vessels, skin epithelial cell secretions in eccrine sweat glands, colonic interstitial cells, liver sinusoids and interstitial cells, and cells in the central nervous system (CNS), such as microglial cells in brain white matter (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 its specific ligands distributed either on the same cell (cis-binding) or on different cells (trans-binding). CD22 cis-binding is generally found between adjacent molecules, where the glycan binding site of the CD22 molecule is ligated to the glycan molecule of another CD22 or glycoprotein on the same cell to form homo-oligomers or homo-multimers. It has been demonstrated that CD22 cis-binding exerts a masking effect on the molecule, preventing Siglecs from forming cell-cell ligations (trans-binding). In resting B cells, CD22 is the predominant cis-ligand for itself, forming CD22 homo-oligomers.Trans-binding of CD22 results in physical association of Siglecs with the B cell receptor (BCR) to exert the maximal inhibitory response required for immune tolerance through the elicitation of inhibitory immunological 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). Further 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. [ka] [ka]

[0048] CD22 is highly expressed on microglial cells, 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 attenuate microglial activation through secretion of IL-10. It has been discovered by the inventors of the present disclosure that certain anti-CD22 antibodies can induce the internalization of CD22 in human microglia. It has also been found that CD22 binds to oligomeric Aβ1-42, and that the internalization of CD22 promotes the clearance of Aβ, both through phagocytosis of CD22-bound Aβ1-42 and macropinocytosis of soluble Aβ. Thus, in some embodiments, methods are provided herein that promote the removal of Aβ using the anti-CD22 antibodies disclosed herein. In some embodiments, methods are provided herein that treat Aβ-related diseases or disorders using the anti-CD22 antibodies disclosed herein. In some embodiments, the Aβ-related disease or disorder is AD.

[0049] As used herein, and as understood in the art, "internalization" of CD22 refers to endocytosis of CD22, which is the process by which a cell invaginates and engulfs a CD22 molecule. There are three types of endocytosis processes: pinocytosis, phagocytosis, and receptor-mediated endocytosis. Pinocytosis, also called "cell-drinking," is the process by which a cell engulfs a small amount of extracellular fluid (along with any small particles that may be present) into the cell through a process of invagination. Phagocytosis is the process by which a relatively large molecule or organism (e.g., a bacterial cell) is engulfed by a cell. Receptor-mediated endocytosis is the process by which material binds directly onto receptor proteins on the cell membrane, initiating the process of invagination and the formation of a protein coating layer known as a clathrin-coat. The vesicles formed in receptor-mediated endocytosis contain this clathrin protein coating. CD22 on the cell membrane is internalized through constitutive clathrin-mediated endocytosis. Upon entering the endosomal compartment, the low pH environment releases it from binding to its specific ligand (eg, CD22 bound in cis) and the free CD22 returns to the cell surface for cis- or trans-ligand binding.

[0050] Current anti-Aβ antibody treatments for AD rely heavily on FcγR-mediated phagocytosis to reduce amyloid burden and are associated with undesirable side effects. Specifically, magnetic resonance imaging (MRI) abnormalities, such as signal changes indicative of "vasogenic edema" (VE) and microhemorrhages (mH), have been observed in a dose-dependent manner in patients administered amyloid-modifying therapeutics (anti-Aβ antibodies). Amyloid-associated imaging abnormalities (ARIA) have been proposed by the Alzheimer's Association Research Roundtable Workgroup to describe vasogenic edema (ARIA-E) and microhemorrhages and hemosiderin deposits (ARIA-H). ARIA-E is primarily caused by inflammation and is tightly linked to mAbs that bind to fibrillar forms of Aβ. FcγR-mediated inflammation in microglia may play a major role. For example, inflammation mediated by FcγR-induced proinflammatory cytokine release exacerbates damage to vascular integrity. Crenzumab, a humanized IgG4 antibody that targets all forms of Aβ, is associated with fewer ARIA-E events because the IgG4 Fc region reduces association to microglial FcγRs compared to, for example, IgG1 Fc.

[0051] Thus, the anti-CD22 antibodies provided herein provide a novel mechanism of Aβ clearance that is not only highly efficient but also associated with reduced vascular side effects such as ARIA-E and / or ARIA-H. Unlike Aβ-targeting antibodies, the anti-CD22 antibodies disclosed herein promote Aβ clearance via CD22 internalization that does not involve cross-linking of therapeutic antibodies with FcγR, thereby avoiding subsequent pro-inflammatory responses. Thus, a method of efficient Aβ clearance with reduced vascular side effects is provided herein. In some embodiments, the methods provided herein do not cause vascular side effects.

[0052] The anti-CD22 antibodies disclosed herein (e.g., SM03 or SM06) (a) promote cis-trans conversion of CD22 and / or (b) induce internalization of CD22. The anti-CD22 antibodies disclosed herein promote cis-trans conversion of CD22 by disrupting the cis-binding of CD22 on the surface of B cells or microglial cells and accelerating the internalization of surface CD22. When CD22 molecules are internalized with an antibody that disrupts the cis-binding of homo-clusters, the CD22 molecules are recycled to the cell surface together with the antibody, where the antibody sterically hinders further cis-binding, thereby promoting the trans-binding of recycled CD22. Thus, the anti-CD22 antibodies disclosed herein can also promote immune tolerance in both the central and peripheral immune systems by disrupting the cis-binding of CD22 on B cells, B cell-derived cell lines and microglia, and promoting the cis-trans conversion of 2,6-sialic acid binding in self-tissue, which allows the immune tolerance of self-tissue to be regained and attenuates pro-inflammatory cytokine release. By promoting the trans-binding of CD22 to neuronal 2,6-sialic acid, the anti-CD22 antibodies disclosed herein not only inhibit synaptic phagocytosis and neuronal death, but also suppress NF-κB signaling and interleukin-6 (IL-6) secretion in microglial cells. Thus, the methods disclosed herein have the additional therapeutic benefit of reducing neuroinflammation. Thus, in some embodiments, methods are provided herein for reducing neuroinflammation using the anti-CD22 antibodies disclosed herein. In some embodiments, methods are provided herein for treating neuroinflammation-related diseases or disorders using the anti-CD22 antibodies disclosed herein.

[0053] Provided herein is a method of promoting the removal of Aβ plaques in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. Also provided herein is a method of 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 specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. In some embodiments, the subject is a human. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD22. In some embodiments, the Aβ-related disease or disorder is AD. In some embodiments, the Aβ-related disease or disorder is preclinical AD.

[0054] In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for removing Aβ plaques. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for the preparation of a medicament for removing Aβ plaques. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 in the treatment of an Aβ-related disease or disorder. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for the preparation of a medicament for the treatment of an Aβ-related disease or disorder. In the above uses, the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. In some embodiments, the Aβ-related disease or disorder is AD. In some embodiments, the Aβ-related disease or disorder is preclinical AD. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD22.

[0055] Treatment with the CD22 antibody disclosed herein increased the rate of Aβ internalization by 5.86×10 as measured using ELISA of Aβ-treated microglial cell lysates. -6 pg / s / cell (data not shown). In some embodiments of the uses or methods provided herein, Aβ was 5.86×10 -6 It is removed at a rate of pg / s / cell.

[0056] The method provided herein can treat Aβ-related disease or disorder.Aβ-related disease or disorder can be clinical or preclinical AD, prodromal AD, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disorder caused by pathogenic Aβ peptide in blood vessels.

[0057] In some embodiments, the methods provided herein can be used to treat AD. As described above and well known in the art, removal of oligomeric Aβ is known to provide clinical benefit in 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.

[0058] In some embodiments, the methods provided herein may be used to treat Down's syndrome. In some embodiments, the methods provided herein may be used to treat clinical or preclinical CAA. In some embodiments, the methods provided herein may be used to treat Parkinson's disease. In some embodiments, the methods provided herein may be used to treat multi-infarct dementia. In some embodiments, the methods provided herein may be used to treat cerebral amyloid angiopathy. In some embodiments, the methods provided herein may be used to treat glaucoma. In some embodiments, the methods provided herein may be used to treat pre-eclampsia. In some embodiments, the methods provided herein may be used to treat cognitive impairment. In some embodiments, the methods provided herein may be used to treat memory loss. In some embodiments, the methods provided herein may be used to treat vascular disorders caused by pathogenic Aβ peptides in blood vessels.

[0059] Also provided herein is a method of reducing neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. Also provided herein is a method of treating a disease or disorder associated with neuroinflammation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. In some embodiments, the methods provided herein prevent synaptic phagocytosis and neuronal death. In some embodiments, the methods provided herein prevent synaptic phagocytosis and neuronal death by at least 20%, at least 30, at least 40%, at least more than 50%, and at least more than 60%. In some embodiments, the methods provided herein prevent synaptic phagocytosis 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.

[0060] In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for reducing neuroinflammation. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for the preparation of a medicament for reducing neuroinflammation. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 in the treatment of a disease or disorder associated with neuroinflammation. In some embodiments, provided herein is the use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 for the preparation of a medicament for the treatment of a disease or disorder associated with neuroinflammation. In the above uses, the antibody or antigen-binding fragment (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD22.

[0061] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods described herein promote cis-trans conversion of CD22. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods described herein induce internalization of CD22. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods described herein promote cis-trans conversion of CD22 and induce internalization of CD22.

[0062] As used herein, the term "treat" and its grammatical equivalents refer to the action of suppressing, eliminating, reducing, and / or ameliorating the symptoms, severity of symptoms, and / or frequency of symptoms associated with the disease or disorder being treated, in relation to a disease or condition, or a subject with a disease or condition.For example, when used in reference to AD, the term "treat" and its grammatical equivalents refer to the action of reducing the severity of disease or slowing or slowing the progression of disease, including but not limited to (a) reducing the amount of Aβ plaque or slowing the rate of pathological Aβ accumulation, or (b) delaying, ameliorating, or minimizing one or more symptoms associated with AD, such as dementia or cognitive impairment.

[0063] As used herein, the term "administer" and its grammatical equivalents refer to the act of delivering or causing to be delivered a therapeutic agent or pharmaceutical composition to the body of a subject by methods described herein or otherwise known in the art. The therapeutic agent may be a compound, a polypeptide, or a cell. Administering a therapeutic agent or pharmaceutical composition includes formulating the therapeutic agent or pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM) or intraperitoneal (IP); subcutaneous (SC), transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

[0064] As used herein, the terms "effective amount", "therapeutically effective amount" and their grammatical equivalents refer to the administration of an agent to a subject in an amount capable of having any detectable positive effect on any symptom, aspect or feature of a disease, disorder or condition when administered to a subject, either alone or as part of a pharmaceutical composition, and either in a single dose or as part of a series of doses. A therapeutically effective amount can be determined by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, depending on the age, weight and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. A therapeutically effective amount is also an amount such that any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. The appropriate "effective amount" in any individual case may vary according to factors such as the disease state, age, sex and weight of the individual, and can be determined by one of ordinary skill in the art using routine experimentation. A "prophylactically effective amount" refers to an amount effective at the dosages and for the period of time necessary to achieve the desired prophylactic result, e.g., delaying or preventing the onset of a disease or disorder. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will generally be less than the therapeutically effective amount.

[0065] The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, dogs, cats, rodents, etc., that is the recipient of a particular treatment. The subject may be a human. The subject may be a patient with a particular disease. 5.2 CD22 antibody

[0066] As described in the above section, methods and uses of antibodies or antigen-binding fragments thereof that specifically bind to CD22 are provided herein. The term "antibody" and its grammatical equivalents, as used herein, refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination of the above, via at least one antigen-binding site, which is usually located in the variable region of the immunoglobulin molecule. As used herein, this term encompasses intact polyclonal antibodies, intact 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) that contain antigen-binding sites, so long as the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse, rabbit, camel, primate, chimeric, humanized and human antibodies. Antibodies may 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), based on the identity of their heavy chain constant domains, called alpha, delta, epsilon, gamma and mu, respectively. In some embodiments, an antibody may comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.

[0067] Unless expressly indicated otherwise, the term "antibody" as used herein includes "antigen-binding fragments" of intact antibodies. The term "antigen-binding fragment" as used herein refers to a portion or fragment of an intact antibody that is an antigen-determining variable region of the intact antibody. Examples of antigen-binding fragments include Fab (a monovalent fragment consisting of the VL, VH, CL and CH1 domains without the hinge region), Fab' (a monovalent fragment consisting of the VL, VH, CL and CH1 domains joined at the hinge region), F(ab')2 (a bivalent fragment containing two Fab fragments linked by a disulfide bridge 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., a single polypeptide chain having the VL and VH domains joined by recombinant means). These include, but are not limited to, bispecific or multispecific antibodies formed from antibody fragments, such as scFvs (heavy chain antibodies), heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), disulfide-linked scFvs (dsscFvs), diabodies (bivalent bispecific antibodies), tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVDs), single variable domain antibodies (sdAbs or dAbs; e.g., camelid antibodies, alpaca antibodies), and single variable domains of heavy chain antibodies (VHHs), as well as antibody fragments. A "bispecific" antibody is an artificial hybrid antibody that has 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 linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0068] An antibody or its antigen-binding fragment may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antigen-binding fragment with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al. (1995) Human antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine tags to generate 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 papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antigen-binding fragments and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0069] The term "humanized antibody" as used herein refers to a form of a non-human (e.g., murine) antibody that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof that contains minimal non-human sequences. Typically, a humanized antibody is a human immunoglobulin. In some cases, Fv framework region residues of a human immunoglobulin are replaced with corresponding residues in an antibody from a non-human species. In some cases, residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and / or binding capacity. A humanized antibody can be further modified by substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize the antibody specificity, affinity, and / or binding capacity. The term "human antibody" as used herein refers to an antibody produced by a human or an antibody that has an amino acid sequence that corresponds to an antibody produced by a human, made using any of the techniques known in the art.

[0070] The term "heavy chain", when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa, the amino-terminal portion of which contains a variable region (VH) of about 120-130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. In some embodiments, the heavy chain constant region is composed of three domains: CH1, CH2, and CH3, with a short flexible hinge region connecting the CH1 and CH2 domains. The constant region can be one of five distinct types, called alpha (a), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains give rise to the five known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chains can be human heavy chains.

[0071] The term "light chain", when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The light chain constant region is composed of one domain, CL. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, called kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

[0072] The term "variable domain" or "variable region" refers to a portion of an antibody light or heavy chain that is generally located at the amino terminus of the light or heavy chain, has a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, and is used in the binding and specificity of each particular antibody to its particular antigen. Variable domains vary widely in sequence among different antibodies. The variability in sequence is concentrated in the CDRs, while the less variable portions of the variable domains are called framework regions (FRs). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. In some embodiments, each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The numbering of amino acid positions used herein is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (US Department of Health and Human Services, Washington, DC) 5th ed.

[0073] CDR refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of an antibody VL β-sheet framework. Thus, CDR is a variable region sequence interspersed within a framework region sequence. CDR regions are well known to those skilled in the art and have been defined by various methods / systems. These systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM and Contact. For example, Kabat defines the most hypervariable region within an antibody variable (V) domain (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 location of structural loop regions and defines CDR region sequences as residues that are not part of the conserved β-sheet framework and therefore can adopt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terminologies are well recognized in the art. Furthermore, the IMGT system is based on sequence variability and location within the structure of the variable region. 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 analysis of antibody sequences and determination of CDRs. The positions of the CDRs within canonical antibody variable domains have been determined by comparison of numerous structures (Al-Lazikani et al, J. Mol. Biol. 273:927-948 (1997); Morea et al, Methods 20:267-279 (2000)).Because the numbers of residues within hypervariable regions vary in different antibodies, additional residues compared to the canonical position are conventionally numbered a, b, c, etc. next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is likewise well known to those of skill in the art.

[0074] For example, CDRs defined according to either the Kabat (hypervariable) or Chothia (structural) designations are shown in the table below. [Table 5] 1 Residue numbering follows the nomenclature of Kabat et al., supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra.

[0075] One or more CDRs may also be incorporated into a molecule, either covalently or non-covalently, to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) non-covalently. The CDRs allow the immunoadhesin to bind to a specific antigen of interest.

[0076] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to a site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, e.g., a localized region on the surface of an antigen. Target molecules can include proteins, peptides, nucleic acids, carbohydrates, or lipids. An epitope with immunogenic activity is a portion of a target molecule that elicits 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, as determined by any method known in the art, including, for example, by immunoassay. An antigenic epitope is not necessarily immunogenic. Epitopes often consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. The term "epitope" includes linear epitopes and conformational epitopes. The region of a target molecule (e.g., a polypeptide) that contributes to an epitope can be contiguous amino acids of the polypeptide, or the epitope can be derived from two or more discontinuous regions of the target molecule together. An epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding (also called conformational epitopes) are typically lost upon protein denaturation. An epitope typically comprises at least 3, more usually at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

[0077] The term "specifically binds" as used herein means that a polypeptide or molecule interacts with an epitope, protein or target molecule more frequently, more rapidly, for a longer duration, with a higher affinity, or some combination of the above, than with alternative substances, including related and unrelated proteins. Binding moieties (e.g., antibodies) that specifically bind to a target molecule (e.g., an antigen) can be identified, for example, by immunoassays, ELISA, biolayer interferometry ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a specific response is at least two times the background signal or noise, and may be greater than 10 times the background. See, for example, Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 for a discussion of antibody specificity. A binding moiety that specifically binds to a target molecule can bind to the target molecule with a higher affinity than its affinity for a different molecule. 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 a different molecule with such low affinity that the binding is not detectable using assays described herein or otherwise known in the art. In some embodiments, "specifically binds" refers to, for example, a binding moiety that has a K D In some embodiments, "specifically binds" means that a polypeptide or molecule binds to a molecular target with a K of about 10 μM or less, or about 1 μM or less. DIn some embodiments, "specifically binds" means that a polypeptide or molecule binds to a target with a K of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. D Specific binding means binding to a target at a specific site. Due to sequence identity between homologous proteins in different species, specific binding may include a polypeptide or molecule that recognizes a protein or target in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding may include a polypeptide or molecule that recognizes more than one protein or target. It is understood that in some embodiments, a binding moiety (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, i.e., binding to a single target (although it may include exclusive binding). Thus, a binding moiety (e.g., an antibody) can, in some embodiments, specifically bind to more than one target. For example, an antibody may, in certain instances, contain two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be bispecific and contain at least two antigen binding sites with different specificities.

[0078] The term "binding affinity," as used herein, generally refers to the strength of the total non-covalent interactions between a binding moiety and a target molecule (e.g., an antigen). Binding between a binding moiety and a target molecule is a reversible process, and the affinity of binding is typically measured by the equilibrium dissociation constant (K D ) is reported as K D is the dissociation rate (k off or k d ) association rate (k on or k a ) of the binding pair. D The lower the K, the higher the affinity. A is the equilibrium association constant, which is the reciprocal of the equilibrium dissociation constant, i.e., =1 / K DRegarding antibody-antigen interactions, K D can be calculated as the ratio of the product of the concentration of free antibody and the concentration of free antigen to the concentration of the antibody-antigen complex, ie, [antigen] x [antibody] / [antigen-antibody].

[0079] A variety of methods for measuring binding affinity are known in the art, 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 "K value" can be measured by assays known in the art, for example, by binding assays. D can be measured by radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293:865-881). D or K D Values ​​can also be measured by using 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 using a surface plasmon resonance assay by using a BIAcore system (eg, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0080] The term "variant," as used herein with respect to a protein or polypeptide having a particular 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, etc.) amino acid substitutions, deletions, and / or additions compared to the reference protein or polypeptide. The changes to the amino acid sequence can be amino acid substitutions. The changes to the amino acid sequence can be conservative amino acid substitutions. A functional fragment or functional variant of a protein or polypeptide maintains the basic structural and functional properties of the reference protein or polypeptide.

[0081] The terms "polypeptide", "peptide", "protein" and their grammatical equivalents, as used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched. It may contain non-natural or modified amino acids or may be interrupted by non-amino acids. Polypeptides, peptides or proteins may also be modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

[0082] The terms "polynucleotide", "nucleic acid" and their grammatical equivalents, when used interchangeably herein, refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, 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.

[0083] As used herein, the term "encode" and its grammatical equivalents refer to the inherent property of a particular sequence of nucleotides in a polynucleotide or nucleic acid, e.g., a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Protein and RNA encoding nucleotide sequences may include introns.

[0084] An "isolated" polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition is a polypeptide, peptide, protein, antibody, polynucleotide, vector, cell or composition in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer present in the 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.

[0085] The terms "identical", percent "identity" and their grammatical equivalents, when used herein with respect to two or more polynucleotides or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence (with gaps introduced as necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignment are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variants. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning 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 compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of the amino acid sequence that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues long, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 residues, e.g., at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies.In some embodiments, the identity exists over 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 in length, or any integer value therebetween. In some embodiments, the identity exists over a region longer than 60-80 bases, e.g., over a region of at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., nucleotide sequences encoding a protein of interest.

[0086] "Conservative amino acid substitution", as used herein, refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. "Conservatively similar" amino acid or residue, as used herein, refers to non-identical amino acid residues having similar side chains. Families of amino acid residues having similar side chains are 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0087] The term "vector" and its grammatical equivalents, as used herein, refers to a vehicle used to carry genetic material (e.g., polynucleotide sequences) that can be introduced into a host cell, where it can be replicated and / or expressed. Applicable vectors for use include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include, for example, selection sequences or markers operable for stable integration into a host cell chromosome. In addition, the vector can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients that are not in the culture medium. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more polynucleotides are co-expressed, both polynucleotides can be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the coding polynucleotide can be operably linked to one common expression control sequence, or can be linked to different expression control sequences, for example, one inducible promoter and one constitutive promoter.The introduction of polynucleotide into host cell can be confirmed using methods well known in the art.It is understood by those skilled in the art that polynucleotide is expressed in sufficient amounts to produce desired product (e.g., anti-CD22 antibody or antigen-binding fragment), and it is further understood that expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0088] As used herein, the term "host cell" refers to a cell into which genetic material, such as a recombinant expression vector, can be or has been introduced. Host cells include not only the subject cell into which exogenous genetic material has been introduced, but also the progeny of such a cell. Such progeny may not be identical to the parent cell, since certain modifications may occur in subsequent generations, either due to mutations or environmental influences.

[0089] As used herein, as understood in the art, "EC" refers to the effective concentration of a drug (e.g., an antibody) and is commonly used in dose-response curves. The "effect" of a drug can be a positive (activating) effect or a negative effect. The term "EC50" refers to the concentration of an active drug (e.g., an antibody) that gives a half-maximal response. Also as used herein, as understood in the art, "IC" refers to the concentration of a drug that has an inhibitory effect and is also commonly used for dose-response curves. The term "IC50" refers to the concentration of a drug (e.g., an antibody) at which the activity it inhibits is reduced by half. 5.2.1 Exemplary CD22 Antibodies

[0090] As described in the above section, the method and use of an antibody or antigen-binding fragment thereof that specifically binds to CD22 (a) promotes cis-trans conversion of CD22 and / or (b) induces internalization of CD22 are provided herein. The antibody and antigen-binding fragment provided herein can be used in Aβ removal, reducing neuroinflammation, or both. The antibody and antigen-binding fragment provided herein can also be used in the treatment of diseases or disorders associated with Aβ and / or neuroinflammation. In some embodiments, the antibody used in the method provided herein specifically binds to human CD22.

[0091] In some embodiments, the anti-CD22 antibody that may be used in the methods disclosed herein is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. In certain embodiments, the antibody comprises a heavy chain constant region, e.g., an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, or any of the above constant regions with glycoforms at glycosylation sites and / or modified glycosylation sites.

[0092] In some embodiments, an antigen-binding fragment of an anti-CD22 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), a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv) or a (scFv)2. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a single domain antibody (sdAb). In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a heavy chain antibody (HCAb). In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a Fab. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a Fab'. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a F(ab')2. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is an Fv. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a scFv. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a disulfide-linked scFv [(scFv)2]. In some embodiments, the antigen-binding fragment of an anti-CD22 antibody is a diabody (dAb).

[0093] In some embodiments, recombinant anti-CD22 antibodies or antigen-binding fragments are used in the methods disclosed herein. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise camelid (e.g., camel, dromedary, and llama) antibodies or antigen-binding fragments. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, an anti-CD22 humanized scFv is provided herein. In some embodiments, an anti-CD22 human humanized Fab is provided herein.

[0094] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods provided herein are isolated. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods provided herein are substantially pure.

[0095] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods provided herein comprise a monovalent antigen-binding site. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments comprise a monospecific binding site. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments comprise a bivalent binding site.

[0096] In some embodiments, monoclonal antibodies are modified by using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of a mouse monoclonal antibody are replaced with the constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are shortened or removed to generate the desired antibody fragment of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region(s) is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0097] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is a humanized antibody or antigen-binding fragment. Various methods for generating humanized antibodies are known in the art. Methods for achieving high affinity binding with humanized antibodies are known in the art. A non-limiting example of such a method is hypermutation of the variable region and selection of cells expressing such high affinity antibodies (affinity maturation). In addition to using a display library, the identified antigen (e.g., recombinant CD22 or an epitope thereof) can be used to immunize a non-human animal, e.g., a rodent. In certain embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and / or disclosed herein. In some embodiments, mice can be immunized with antigen (e.g., recombinant CD22 or an epitope thereof).

[0098] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is a human antibody or antigen-binding fragment. Human antibodies can be prepared using a variety of techniques known in the art. In some embodiments, human antibodies are generated in vitro from immunized immortalized human B lymphocytes. In some embodiments, human antibodies are generated from lymphocytes isolated from immunized individuals. In either case, cells producing antibodies against the target antigen can be generated and isolated. In some embodiments, human antibodies are selected from a phage library, if the phage library expresses human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for generating and using antibody phage libraries are well known in the art. Once an antibody is identified, affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis, can be used to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice that contain human immunoglobulin loci and that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0099] The specific CDR sequences defined herein are generally based on a combination of Kabat and Chothia definitions.However, it is understood that the reference to the heavy chain CDR(s) and / or light chain CDR(s) of a specific antibody encompasses all CDR definitions known to those skilled in the art.The anti-CD22 antibody or antigen-binding fragment that can be used in the methods provided herein includes SM03 and SM06, and their variations and derivatives.

[0100] The term "SM03" refers to a chimeric antibody against human CD22 (hCD22). The sequence characteristics of SM03 are provided in the following table. Further description of the structural and functional characteristics of SM03 can be found, for example, in Yang et al. (2006), Chinese J New Drug 15(3):186-92; and Chinese Patent No. ZL03123054.7, which are incorporated herein by reference in their entirety.

[0101] The term "SM06" refers to a framework patch or humanized version of the chimeric antibody SM03 that has been re-engineered to reduce its potential immunogenicity and exhibits affinity and specificity for hCD22. The sequence features of SM06 are provided in the table below. Further description of the structural and functional features of SM06 can be found, for example, in Liang et al. (2006) Chinese J New Drug 15(21):1832-36; Chinese Patent No. ZL01144894.6 and U.S. Patent No. 7,321,026 B2 and U.S. Patent No. 7,338,659 B2, which are incorporated herein by reference in their entirety. Table 1: CDR sequences of SM03 and SM06 [Table 1] Table 2: Variable regions of SM03 and SM06 [Table 2]

[0102] In some embodiments, anti-CD22 antibodies or antigen-binding fragments that may be used in the methods provided herein comprise one, two, three, four, five, and / or six CDRs of SM03 / SM06. In some embodiments, anti-CD22 antibodies or antigen-binding fragments comprise a VL that comprises one, two, and / or three VL CDRs from Table 1. In some embodiments, anti-CD22 antibodies or antigen-binding fragments provided herein comprise a VH that comprises one, two, and / or three VH CDRs from Table 1. In some embodiments, anti-CD22 antibodies or antigen-binding fragments provided herein comprise one, two, and / or three VL CDRs and one, two, and / or three VH CDRs from Table 1.

[0103] In some embodiments, an antibody or antigen-binding fragment thereof that may be used in the methods provided herein comprises a light chain variable region (VL) comprising: (1) a light chain CDR1 (VL CDR1) having the amino acid sequence of SEQ ID NO: 1; (2) a light chain CDR2 (VL CDR2) having the amino acid sequence of SEQ ID NO: 2; or (3) a light chain CDR3 (VL CDR3) having the 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 the amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 2; and (3) a VL CDR3 having the 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 CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions and / or deletions in the VL CDRs.

[0104] 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 (VH CDR1) having the amino acid sequence of SEQ ID NO: 4; (2) a heavy chain CDR2 (VH CDR2) having the amino acid sequence of SEQ ID NO: 5; or (3) a heavy chain CDR3 (VH CDR3) having the 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 up to 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 the amino acid sequence of SEQ ID NO: 4; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 5; and (3) a VH CDR3 having the 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 CDRs. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[0105] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds CD22, comprising: (a) a VL comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 1; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 2; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 3; or a variant thereof having up to about five amino acid substitutions, additions, and / or deletions in the VL CDRs; and (b) a VH comprising: (1) a VH CDR1 having the amino acid sequence of SEQ ID NO: 4; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 5; and (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 6; or a variant thereof having up to about five amino acid substitutions, additions, and / or deletions in the VH CDRs.

[0106] It is well known in the art that VH CDR3 and VL CDR3 domains play an important role in the binding specificity / affinity of an antibody to an antigen. Thus, in some embodiments, an anti-CD22 antibody or antigen-binding fragment thereof that can be used in the methods disclosed herein can have suitable association / dissociation kinetics with human CD22 and can have VH CDR3 and VL CDR3 structurally identical or related to those of SM03 and / or SM06. The consensus motif for SM03 VL CDR3, which includes the amino acid sequence: QQGNTLPWT (SEQ ID NO: 3), can be modified by substituting one or more amino acids to adjust the antibody affinity without changing its binding specificity, or alternatively, can be replaced by the VL CDR3 of an unrelated human antibody that shows sufficient similarity to SM03 VL CDR3, using the criteria described in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Similarly, the consensus motif for SM03 VH CDR3, which comprises the amino acid sequence: HSGYGSSYGVLFAY (SEQ ID NO:6), may be modified by substituting one or more amino acids to adjust the antibody affinity without altering its binding specificity, or alternatively, may be replaced by the VH CDR3 of an unrelated human antibody that shows sufficient similarity to SM03 VH CDR3, using the criteria set forth in Chinese Patent No. ZL200880024788.2, which is incorporated herein by reference. Those skilled in the art will appreciate that certain substitution(s) of amino acids within the CDR3 domain, particularly substitutions with conservative amino acids, do not alter the epitope specificity of the antibody.Thus, in some embodiments, the CDR3 of an antibody or antigen-binding fragment provided herein (e.g., SM03 or SM06) is: (1) identical in number of residues and exhibits 50% or greater sequence homology to the SM03 CDR3; (2) contains at least one, and preferably more, aromatic residues that are identical or conservatively similar to the residue(s) at the corresponding position(s) in the SM03 CDR3; (3) contains at least one, and preferably more, charged residues that are identical or conservatively similar to the residue(s) at the corresponding position(s) in the SM03 CDR3; and / or (4) contains at least one, and preferably more, charged residues that are identical or conservatively similar to the residue(s) at the corresponding position(s) in the SM03 CDR3 at positions known to be important for maintaining the binding site structure / contacts of anti-CD22 antibodies as determined by crystal structure and / or computer database analysis. The SM03 VL and / or VH CDR3 domains may be replaced with a CDR3 from a human or primate antibody that contains at least one, and preferably more, amino acid residues that are identical or conservatively similar to the residue(s) at the corresponding position(s) in the CDR3 (see Chinese Patent No. ZL200880024788.2, incorporated herein by reference). In some embodiments, no more than one to five conservative amino acid substitutions are made within the SM03 VL and / or VH CDR3 domains, or a VL and / or VH CDR3 from an unrelated primate or human antibody containing no more than one to five conservatively similar residues is used to replace the VL and / or VH CDR3 of SM03 or SM06. In some embodiments, no more than one to three conservative amino acid substitutions are made within the SM03 VL and / or VH CDR3 domains, or a VL and / or VH CDR3 from an unrelated primate or human antibody containing no more than one to three conservatively similar residues is used to replace the VL and / or VH CDR3 of SM03 or SM06.

[0107] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0108] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 85% sequence identity to SEQ ID NO:7. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 90% sequence identity to SEQ ID NO:7. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 95% sequence identity to SEQ ID NO:7. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 98% sequence identity to SEQ ID NO:7. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL having the amino acid sequence of SEQ ID NO:7.

[0109] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 85% sequence identity to SEQ ID NO:8. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 90% sequence identity to SEQ ID NO:8. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 95% sequence identity to SEQ ID NO:8. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 98% sequence identity to SEQ ID NO:8. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH having the amino acid sequence of SEQ ID NO:8.

[0110] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9; and (b) a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:10.

[0111] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:9. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 85% sequence identity to SEQ ID NO:9. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 90% sequence identity to SEQ ID NO:9. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 95% sequence identity to SEQ ID NO:9. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VL that has at least 98% sequence identity to SEQ ID NO:9. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL having the amino acid sequence of SEQ ID NO:9.

[0112] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22 comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof has a VH that has at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VH having the amino acid sequence of SEQ ID NO:10.

[0113] 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 and VH have the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the VL and VH have the amino acid sequences of SEQ ID NOs: 9 and 10, respectively.

[0114] An anti-CD22 antibody or antigen-binding fragment thereof may comprise any VL disclosed herein in combination with any VH disclosed herein. In some embodiments, the 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: 19). In some embodiments, the linker has an amino acid sequence of GSAGSAAGSGEF (SEQ ID NO: 38). In some embodiments, the linker has an amino acid sequence of KESGSVSSEQLAQFRSLD (SEQ ID NO: 39). In some embodiments, the linker has an amino acid sequence of EGKSSGSGSESKS (SEQ ID NO: 40). In some embodiments, the linker has an amino acid sequence of SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 41). In some embodiments, the linker has an amino acid sequence of GGGGGG (SEQ ID NO: 42). 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:20). In some embodiments, the linker has an amino acid sequence of (PA)nP, n=1, 2, 3, 4 or 5 (SEQ ID NO:21).

[0115] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising: (a) a VL comprising VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 7 or 9; and / or (b) a VH comprising VH CDR1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 8 or 10. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising: (a) a VL comprising VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 9; and / or (b) a VH comprising VH CDR1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 10.

[0116] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to CD22, comprising a VL, wherein the VL comprises VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VL comprises VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 9.

[0117] 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 CDR1, 2 and 3 from a VH having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VH comprises VH CDR1, 2 and 3 from a VH having the amino acid sequence of SEQ ID NO: 10.

[0118] 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 the amino acid sequence of SEQ ID NO:7, and the VH comprises a VH CDR1, CDR2 and CDR3 from a VH having the amino acid sequence of SEQ ID NO:8. In some embodiments, the VL comprises a VL CDR1, CDR2 and CDR3 from a VL having the amino acid sequence of SEQ ID NO:9, and the VH comprises a VH CDR1, CDR2 and CDR3 from a VH having the amino acid sequence of SEQ ID NO:10.

[0119] In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein is an antibody designated SM03. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VL from SM03. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VH from SM03. The anti-CD22 antibody or antigen-binding fragment thereof provided herein may have both a VL and a VH from SM03. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VL that includes VL CDR1, 2, and 3 from the VL from SM03. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VH that includes VH CDR1, 2, and 3 from the VH from SM03. The anti-CD22 antibody or antigen-binding fragment thereof provided herein may have a VL that includes VL CDR1, 2, and 3 and a VH that includes VH CDR1, 2, and 3 from the VL and VH, respectively, of SM03. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments thereof provided herein are variants of SM03. The SM03 variants may have a VL that is 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 SM03 variants may have a VH that is 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 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 CDRs. In some embodiments, the variants of SM03 have up to about 5 conservative amino acid substitutions. In some embodiments, the variants of SM03 have up to 3 conservative amino acid substitutions.

[0120] In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein is an antibody designated SM06. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VL from SM06. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VH from SM06. The anti-CD22 antibody or antigen-binding fragment thereof provided herein may have both a VL and a VH from SM06. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VL that includes VL CDR1, 2, and 3 from the VL from SM06. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein has a VH that includes VH CDR1, 2, and 3 from the VH from SM06. The anti-CD22 antibody or antigen-binding fragment thereof provided herein may have a VL that includes VL CDR1, 2, and 3 and a VH that includes VH CDR1, 2, and 3 from the VL and VH of SM06, respectively. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof provided herein is a variant of SM06. The SM06 variant may have a VL that is 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 SM06 variant may have a VH that is 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 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 CDRs. In some embodiments, the variant of SM06 has up to about 5 conservative amino acid substitutions. In some embodiments, the variant of SM06 has up to 3 conservative amino acid substitutions.

[0121] In some embodiments, the anti-CD22 antibody or antigen-binding fragment that can be used in the methods disclosed herein comprises a VH or VL having at least one framework (FR) region. The FR region for the VL can be derived from the VK10 mouse germline family. In some embodiments, the FR1, FR2, FR3 and FR4 for the VL can have the amino acid sequences of SEQ ID NOs: 30, 31, 32 and 33, respectively (SM03 VL framework sequences). In some embodiments, the FR region for the VH can be derived from the VH5 mouse germline family. In some embodiments, the FR1, FR2, FR3 and FR4 for the VH can have the amino acid sequences of SEQ ID NOs: 34, 35, 36 and 37, respectively (SM03 VH framework sequences). In some embodiments, the FR1 region for the VL may be from the VκID human germline family, the FR2 region for the VL may be from the Vκ1 human germline family, the FR3 region for the VL may be from the Vκ1 human germline family, and the FR4 region for the VL may be from the VκJ1 human germline family. In some embodiments, the FR1, FR2, FR3, and FR4 for the VL may 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 the VH may be from the VκJ1 human germline family, the FR2 region for the VL may be from the VκJ1 human germline family, the FR3 region for the VL may be from the VκJ1 human germline family, and the FR4 region for the VL may be from the VκJ1 human germline family. In some embodiments, the FR1, FR2, FR3, and FR4 for the VL may have the amino acid sequences of SEQ ID NOs: 22, 23, 24, and 25, respectively (SM06 VL framework sequences). H 3 human germline family; the FR2 region for VH can be H 3 human germline family; the FR3 region for VH can be H 3 human germline family; the FR4 region for VH is H It may be derived from the J5 human germline family. In some embodiments, FR1, FR2, FR3 and FR4 for the VH may have the amino acid sequences of SEQ ID NOs: 26, 27, 28 and 29, respectively (SM06 VH framework sequences).

[0122] The present disclosure further contemplates additional variants and equivalents substantially homologous to the recombinant, monoclonal, chimeric, humanized and human antibodies described herein, or antibody fragments thereof. 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(s), glycosylation, immunogenicity and / or solubility. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

[0123] The variation may be a substitution, deletion or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence compared to the native antibody or polypeptide sequence. In some embodiments, the amino acid substitution is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., replacing a leucine with a serine, e.g., a conservative amino acid replacement. The insertion or deletion may range from about 1-5 amino acids. In some embodiments, the substitution, deletion or insertion comprises fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the parent molecule. In some embodiments, biologically useful and / or relevant variations in amino acid sequence may be determined by systematically making insertions, deletions or substitutions in the sequence and testing the resulting variant proteins for activity compared to the parent protein.

[0124] In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof that may be used in the methods provided herein is a chimeric antibody or antigen-binding fragment. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof that may be used in the methods provided herein is a humanized antibody or antigen-binding fragment. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof comprises a VL CDR1, a VL CDR2, a VL CDR3, a VH CDR1, a VH CDR2 and / or a VH CDR3 from an antibody or antigen-binding fragment described herein. In some embodiments, the anti-CD22 antibody or antigen-binding fragment thereof comprises a variant of the anti-CD22 antibody or antigen-binding fragment described herein. In some embodiments, the variant of the anti-CD22 antibody or antigen-binding fragment comprises 1-30 amino acid substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise 1-25 amino acid substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise 1-20 substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise 1-15 substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise 1-10 substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise 1-5 conservative amino acid substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, the variant of the anti-CD22 antibody or antigen-binding fragment comprises 1-3 amino acid substitutions, additions and / or deletions in the anti-CD22 antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions and / or deletions are conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution(s) are in the CDRs of the antibody or antigen-binding fragment.In some embodiments, the conservative amino acid substitution(s) are not in the CDRs of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution(s) are in the framework regions of the antibody or antigen-binding fragment.

[0125] It is known in the art that the constant region(s) of an antibody mediate several effector functions, and these effector functions may vary depending on the antibody isotype. For example, binding of the C1 component of complement to the Fc region of an IgG or IgM antibody (bound to an antigen) activates the complement system. Complement activation is important in opsonizing and lysing cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind to cells expressing Fc receptors (FcR). There are several Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibodies to Fc receptors on cell surfaces triggers many important and diverse biological responses, including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgA antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgD antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgE antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgM antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG1 antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG2 antibody.In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG3 antibody. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments described herein comprise at least one constant region of a human IgG4 antibody.

[0126] In some embodiments, at least one or more of the constant regions are modified or deleted in the anti-CD22 antibodies or antigen-binding fragments described herein. In some embodiments, the antibodies include modifications to 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 modifications to the constant region include 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 is removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically afforded by the absent 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 domain.

[0127] In some embodiments, the anti-CD22 antibody or antigen-binding fragment comprises an Fc region. In some embodiments, the Fc region is fused via 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 skilled in the art. In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the modified antibodies (e.g., modified Fc regions) provide altered effector functions that in turn affect the biological profile of the antibody. For example, in some embodiments, the Fc regions of the antibodies or antigen-binding fragments provided herein are modified to enhance their ability to cross the blood-brain barrier (BBB). In some embodiments, deletion or inactivation (via point mutation or other means) of the constant region reduces Fc receptor binding of the modified antibody as it circulates. In some embodiments, the constant region modification reduces the immunogenicity of the antibody. In some embodiments, the constant region modification increases the serum half-life of the antibody. In some embodiments, the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications reduce or eliminate ADCC and / or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, specific amino acid substitutions in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduce effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., an "effector-less" antibody). In some embodiments, the antibody does not have ADCC activity and / or does not have CDC activity. In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector function(s). In some embodiments, the constant region modifications increase or enhance ADCC and / or CDC of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties.In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide one or more cytotoxins, oligosaccharides or carbohydrate binding sites. In some embodiments, the anti-CD22 antibody or antigen-binding fragment comprises a variant Fc region engineered by substitution at specific amino acid positions compared to the native Fc region. In some embodiments, the anti-CD22 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of 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.

[0128] In some embodiments, the variants may include addition of amino acid residues at the amino and / or carboxyl termini of the antibody or polypeptide. The length of the additional amino acid residues may range from 1 residue to 100 or more residues. In some embodiments, the variants include an N-terminal methionyl residue. In some embodiments, the variants include additional polypeptides / proteins (e.g., Fc regions) to create fusion proteins. In some embodiments, the variants may be engineered to be detectable and include detectable labels and / or proteins (e.g., fluorescent tags or enzymes).

[0129] Therapeutic antibodies can be re-engineered to facilitate transport across the BBB via various mechanisms, including, for example, receptor-mediated transcytosis, adsorptive transcytosis, carrier-mediated transport, paracellular transport, and diffusion. Of all, receptor-mediated transcytosis (RMT) has been widely explored to facilitate antibody entry. The use of RMT includes transferrin receptor, insulin receptor, low-density lipoprotein receptor (LDL receptor), CD98, TMEM50A, and other surface receptors on endothelial cells, which can perform transcytosis upon binding. Antibodies that bind to transferrin, insulin receptor, CD98, and TEME50A can initiate transcytosis of the entire complex. In the case of the LDL receptor, binding of apolipoproteins can trigger the transcytosis process. Thus, therapeutic antibodies linked to antibody fragments or apolipoproteins are proposed and validated to enhance BBB crossing in animal models.

[0130] Thus, the anti-CD22 antibodies or antigen-binding fragments provided herein can be engineered to enhance their ability to cross the BBB. In some embodiments, the anti-CD22 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 antibodies or antigen-binding fragments provided herein can be fused to a second antibody or antigen-binding fragment that binds to transferrin. In some embodiments, for example, the anti-CD22 antibodies or antigen-binding fragments can be fused to a transferrin receptor-binding Fab fragment. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein can be fused to a second antibody or antigen-binding fragment that binds to insulin receptor. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein can be fused to a second antibody or antigen-binding fragment that binds to CD98. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein may be fused to a second antibody or antigen-binding fragment that binds to TEME50A. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein are fused to an apolipoprotein.

[0131] Variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0132] In some embodiments, variants of anti-CD22 antibodies or antigen-binding fragments disclosed herein may retain the ability to bind to CD22 to a similar, equal or greater extent than the parent antibody or antigen-binding fragment. In some embodiments, variants 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 in amino acid sequence to the parent antibody or antigen-binding fragment. In certain embodiments, variants of anti-CD22 antibodies or antigen-binding fragments comprise the amino acid sequence of the parent anti-CD22 antibody or antigen-binding fragment 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 with certain physical and / or chemical properties is replaced with another amino acid with the same or similar chemical or physical properties.

[0133] In some embodiments, a variant of an anti-CD22 antibody or antigen-binding fragment comprises the amino acid sequence of a parent antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-CD22 antibody or antigen-binding fragment comprises the amino acid sequence of a parent binding antibody or antigen-binding fragment with one or more non-conservative amino acid substitutions, where the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities (e.g., CD22 binding) of the variant. In certain embodiments, one or more conservative amino acid substitutions and / or one or more non-conservative amino acid substitutions may enhance the biological activity of the variant, such that the biological activity of the functional variant is increased compared to the parent binding moiety.

[0134] In some embodiments, the anti-CD22 antibody or antigen-binding fragment described herein is chemically modified, either manually or by intervention. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques. The anti-CD22 antibody or antigen-binding fragment can include one or more analogs of amino acids, including, for example, unnatural amino acids, as well as other modifications known in the art.

[0135] The anti-CD22 antibodies or antigen-binding fragments of the present disclosure may be analyzed for their physical, chemical and / or biological properties by various methods known in the art. In some embodiments, the anti-CD22 antibodies are tested for their ability to bind to CD22 (e.g., human CD22). Binding assays include, but are not limited to, surface plasmon resonance (e.g., Biacore), ELISA and FACS. In some embodiments, the dissociation constant of the binder (e.g., antibody) to CD22 is the dissociation constant determined by surface plasmon resonance (e.g., BIAcore). In addition, the antibodies may be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality and / or purification efficiency.

[0136] Both SM03 and SM06 have a dissociation constant (K D In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein bind to human CD22 with a dissociation constant (K) of 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. D ) and binds to CD22 (e.g., human CD22). Dis about 20 nM or less. D is about 10 nM or less. D is about 5 nM or less. D is about 2 nM or less. D is about 1.5 nM or less. D is about 1 nM or less. D is about 0.5 nM or less. D is about 0.1 nM or less. D is about 50 pM or less. D is about 10 pM or less.

[0137] In some embodiments, the anti-CD22 antibody or antigen-binding fragment has a K 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. D and binds to CD22 (e.g., human CD22). D In some embodiments, K is in the range of 0.1 to 1 nM. D In some embodiments, K is in the range of 0.5 to 5 nM. D In some embodiments, K is in the range of 1 to 10 nM. D In some embodiments, K is in the range of 1 to 5 nM. D In some embodiments, K is in the range of 5 to 50 nM. D In some embodiments, K is in the range of 10 to 100 nM. D is in the range of 50 to 500 nM.

[0138] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is about 0.8×10 9 M -1 The association constant (K AIn some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein binds to CD22 (e.g., human CD22) at 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 x 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 approximately 1 × 10 12 M -1 or higher A and binds to CD22 (e.g., human CD22). A is about 1 x 10 7 M -1 or higher. In some embodiments, K A is about 5 x 10 7 M -1 or higher. In some embodiments, K A is about 1 x 10 8 M -1 or higher. In some embodiments, K A is about 5 x 10 8 M -1 or higher. In some embodiments, K A is about 8 x 10 8 M -1 or higher. In some embodiments, KA is about 1 x 10 9 M -1 or higher. In some embodiments, K A is about 5 x 10 9 M -1 or higher. In some embodiments, K A is about 1 x 10 10 M -1 Or higher.

[0139] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is 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 K in the rangeA and binds to CD22 (e.g., human CD22). A is about 1 x 10 6 ~1×10 7 M -1 In some embodiments, K A is about 1 x 10 7 ~1×10 8 M -1 In some embodiments, K A is about 1 x 10 8 ~1×10 9 M -1 In some embodiments, K A is about 5 x 10 8 ~1×10 9 M -1 In some embodiments, K A is about 5 x 10 8 ~5×10 9 M -1 In some embodiments, K A is about 1 x 10 9 ~1×10 10 M -1 is within the range.

[0140] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein has a binding affinity of 0.0685 RU s as determined by SPR (e.g., BIAcore). -1 or less, or 0.0137RU s -1 In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from human CD22 with a kd of about 0.5 RU s or less. -1 or less, approximately 0.2RU s -1 or less, approximately 0.1RU s -1 or less, approximately 0.08RU s -1 or less, approximately 0.06RU s -1 or less, approximately 0.05RU s -1 or less, approximately 0.04RU s -1or less, approximately 0.03RU s -1 or less, approximately 0.02RU s -1 or less, approximately 0.01RU s -1 or less, approximately 0.008RU s -1 or less, approximately 0.005RU s -1 or less, or about 0.001 RU s -1 In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein dissociate from human CD22 with a kd of about 0.5 RU s or less. -1 or less. In some embodiments, the kd is about 0.2 RU s -1 In some embodiments, the kd is about 0.1 RU s -1 In some embodiments, the kd is about 0.08 RU s -1 In some embodiments, the kd is about 0.06 RU s -1 In some embodiments, the kd is about 0.05 RU s -1 In some embodiments, the kd is about 0.02 RU s -1 In some embodiments, the kd is about 0.01 RU s -1 In some embodiments, the kd is about 0.005 RU s -1 Or less.

[0141] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein have a concentration of 0.001 to 0.5 RU s -1 , 0.001~0.1RUs -1 , 0.001~0.05RUs -1 , 0.005~0.5RUs -1 , 0.005~0.1RUs -1 , 0.01~0.5RUs -1 , 0.01~0.1RUs -1 or 0.01~0.05RUs -1In some embodiments, the kd is in the range of 0.005 to 0.05 RU s -1 In some embodiments, the kd is in the range of 0.01 to 0.1 RU s -1 In some embodiments, the kd is in the range of 0.005 to 0.1 RU s -1 In some embodiments, the kd is in the range of 0.01 to 0.5 RU s -1 In some embodiments, the kd is in the range of 0.01 to 0.1 RU s -1 is within the range.

[0142] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein has a cytoplasmic affinity of 1.13×10 as determined by SPR (e.g., BIAcore). 7 RUS -1 or higher. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein associate with human CD22 with a ka of about 1.0×10 5 RUS -1 or higher, about 5.0×10 5 RUS -1 or higher, about 1.0×10 6 RUS -1 or higher, about 5.0×10 6 RUS -1 or higher, about 1.0×10 7 RUS -1 or higher, about 2.0×10 7 RUS -1 or higher, about 3×10 7 RUS -1 or higher, about 4.0×10 7 RUS -1 or higher, about 5.0×10 7 RUS -1 or higher, about 1.0×10 8 RUS -1 or higher, about 5.0×10 8 RUS-1 or higher, or approximately 1.0 × 10 9 RUS -1 or higher. In some embodiments, the ka is about 1.0×10 6 RUS -1 In some embodiments, the k is about 5.0×10 6 RUS -1 In some embodiments, the k is about 1.0×10 7 RUS -1 In some embodiments, the k is about 2.0×10 7 RUS -1 In some embodiments, the k is about 5.0×10 7 RUS -1 In some embodiments, the k is about 1.0×10 8 RUS -1 In some embodiments, the k is about 1.0×10 9 RUS -1 Or higher.

[0143] In some embodiments, k is 1.0×10 5 ~1.0×10 6 RUS -1 , 1.0×10 6 ~1.0×10 7 RUS -1 , 5.0×10 6 ~5.0×10 7 RUS -1 , 1.0×10 7 ~5.0×10 7 RUS -1 , 1.0×10 7 ~1.0×10 8 RUS -1 or 1.0×10 8 ~1.0×10 9 RUS -1 In some embodiments, ka is in the range of 1.0×10 6 ~1.0×10 7 RUS -1In some embodiments, ka is in the range of 5.0×10 6 ~5.0×10 7 RUS -1 In some embodiments, ka is in the range of 1.0×10 7 ~5.0×10 7 RUS -1 In some embodiments, ka is in the range of 1.0×10 7 ~1.0×10 8 RUS -1 In some embodiments, ka is in the range of 1.0×10 8 ~1.0×10 9 RUS -1 is within the range.

[0144] Epitope mapping is a method to identify the binding site, region or epitope on a target protein to which an antibody binds. A variety of methods are known in the art for mapping epitopes on a target protein. These methods include shotgun mutagenesis, site-directed mutagenesis and alanine scanning; domain or fragment scanning; mutagenesis including but not limited to peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display and ribosome / mRNA display); methods involving proteolysis and mass spectroscopy; and structure determination (e.g., X-ray crystallography and NMR). In some embodiments, the anti-CD22 antibody or antigen-binding fragment described herein is characterized by assays including but not limited to N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion exchange chromatography and papain digestion.

[0145] As described in Leung et al. (2015) Mabs 7(1):66-76; Zhao et al. (2014) PLOS ONE 9(5): e96697; U.S. Pat. No. 9,371,396 B2; and Chinese Patent No. ZL201210286457.4, which are incorporated by reference in their entireties, SM03 and SM06 have a 0.82×10 9 M -1 With an affinity (Ka) in the range of 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 (SEQ ID NO: 18) specifically binds to domain 2 of human CD22 with high affinity. Binding of this conformational epitope induces rapid internalization of CD22 (e.g., 50% of surface CD22 can be internalized within 10 minutes), promotes cis-trans conversion of 2,6-sialic acid bonds of CD22, and can restore immune tolerance. Both SM03 and SM06 also bind to anti-idiotypic antibodies (e.g., LRID) against the antigen binding site (ABS) of SM03 and SM06 with EC50 in the range of 79.9±27.6 ng / ml.

[0146] Thus, the anti-CD22 antibodies or antigen-binding fragments that may be used in the methods disclosed herein can bind to an epitope that does not sterically interfere with the interaction of CD22 with Aβ. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments bind to a specific conformational epitope that does not sterically interfere with the interaction of CD22 with Aβ. In some embodiments, the epitope is 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17). In some embodiments, the epitope comprises 198 VFTRSELKFSPQWSHHGKIVTC 219 (SEQ ID NO: 18). In some embodiments, the epitope is a conformational epitope, 161 CLLNFSCYGYPIQ173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 (SEQ ID NO: 18).

[0147] In some embodiments, the anti-CD22 antibody or antigen-binding fragment that may be used in the methods disclosed herein has a titer of about 0.8×10 9 M -1 K A In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein can bind to this conformational epitope at 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 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 approximately 1 × 10 12 M -1 or higher A In some embodiments, the conformational epitope is bound by K A is about 1 x 10 7 M -1 or higher. In some embodiments, K A is about 5 x 10 7 M -1 or higher. In some embodiments, K A is about 1 x 10 8 M -1 or higher. In some embodiments, KA is about 5 x 10 8 M -1 or higher. In some embodiments, K A is about 8 x 10 8 M -1 or higher. In some embodiments, K A is about 1 x 10 9 M -1 or higher. In some embodiments, K A is about 5 x 10 9 M -1 Or higher.

[0148] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is 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 -1or 5×10 11 ~5×10 12 M -1 K in the range A In some embodiments, the conformational epitope is bound by K A is about 1 x 10 6 ~1×10 7 M -1 In some embodiments, K A is about 1 x 10 6 ~1×10 7 M -1 In some embodiments, K A is about 1 x 10 7 ~1×10 8 M -1 In some embodiments, K A is about 1 x 10 8 ~1×10 9 M -1 In some embodiments, K A is about 5 x 10 8 ~1×10 9 M -1 In some embodiments, K A is about 5 x 10 8 ~5×10 9 M -1 In some embodiments, K A is about 1 x 10 9 ~1×10 10 M -1 is within the range.

[0149] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein bind to anti-idiotypic antibodies with high affinity (e.g., as described in Leung 2015 supra; Zhao 2014 supra). The anti-idiotypic antibodies have a VL and VH each having the amino acid sequences of SEQ ID NOs: 13 and 14, respectively ("LRID"). In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein have an EC of about 0.40 μg / ml or less. 50In some embodiments, the anti-CD22 antibody or antigen-binding fragment binds to LRID with an EC of about 0.08 μg / ml. 50 In some embodiments, the EC 50 is about 5.0 μg / ml, about 2.0 μg / ml, about 1.0 μg / ml, about 0.8 μg / ml, about 0.5 μg / ml, about 0.2 μg / ml, about 0.1 μg / ml, about 0.08 μg / ml, about 0.05 μg / ml, about 0.02 μg / ml, about 0.01 μg / ml, about 0.008 μg / ml, about 0.005 μg / ml, or about 0.001 μg / ml. 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 is approximately 0.01 μg / ml.

[0150] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein have an EC value within the range of 0.01-5 μg / ml, 0.01-1 μg / ml, 0.01-0.5 μg / ml, 0.05-5 μg / ml, 0.05-1 μg / ml, 0.05-0.5 μg / ml, 0.1-5 μg / ml, or 0.1-1 μg / ml. 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC50 is in the range of 0.05 to 0.5 μg / ml.

[0151] In some embodiments, an antibody or antigen-binding fragment that competes with the antibody or antigen-binding fragment provided above for binding to CD22 (e.g., human CD22) is also provided herein. An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other 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 the other antibody to a target, can be determined using known competition experiments, for example, BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments, an anti-CD22 antibody or antigen-binding fragment competes with and inhibits the binding of another antibody or antigen-binding fragment to CD22 (e.g., human CD22) by at least 50%, 60%, 70%, 80%, 90% or 100%. Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: l0.H0l / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0152] In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with SM03 for binding to human CD22. In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with SM06 for binding to human CD22. In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein are radiolabeled I that binds to native CD22 on the human Burkitt's lymphoma cell line Ramos cells. 125-SM03 with an EC of about 5.01 μg / ml or less, or about 1.02 μg / ml or less 50 In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein competes with radiolabeled I that binds to native CD22 on the human Burkitt's lymphoma cell line Ramos cells. 125 -SM03 with an EC of about 5.01 μg / ml or less, or about 1.02 μg / ml 50 In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein competes with radiolabeled I that binds to native CD22 on Ramos cells. 125 -SM03 and an EC of 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. 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is a radiolabeled I antibody that binds to native CD22 on Ramos cells. 125-SM03 and an EC within 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 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 is in the range of about 1 to 5 μg / ml.

[0153] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is a radiolabeled I antibody that binds to native CD22 on microglial cells (e.g., HMC-3 cells). 125 -SM03 and an EC of 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. 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is a radiolabeled I antibody that binds to native CD22 on microglial cells (e.g., HMC-3 cells). 125 -SM03 binding and an EC within 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 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 In some embodiments, the EC 50 is in the range of about 1 to 5 μg / ml.

[0154] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein can induce internalization of about 20%, about 30%, about 40%, about 50%, about 60%, about 70% of surface CD22 within 10 minutes upon contact with a cell (e.g., a B cell or a microglial cell). In some embodiments, the internalization rate is about 30% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 40% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 50% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 60% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 70% of surface CD22 within 10 minutes. In some embodiments, an anti-CD22 antibody or antigen-binding fragment provided herein can induce internalization of about 20%-70%, about 30%-70%, about 40%-70%, about 30%-60%, or about 40%-60% of surface CD22 within 10 minutes upon contact. In some embodiments, the internalization rate is about 20%-70% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 40%-70% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 30%-60% of surface CD22 within 10 minutes. In some embodiments, the internalization rate is about 40%-60% of surface CD22 within 10 minutes.

[0155] In some embodiments, an anti-CD22 antibody or antigen-binding fragment provided herein, upon contact with a cell (e.g., a B cell or a microglial cell), can induce internalization of about 50% of surface CD22 within about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 1 hour. In some embodiments, 50% of surface CD22 is internalized within 2 minutes. In some embodiments, 50% of surface CD22 is internalized within 5 minutes. In some embodiments, 50% of surface CD22 is internalized within 10 minutes. In some embodiments, 50% of surface CD22 is internalized within 15 minutes. In some embodiments, 50% of surface CD22 is internalized within 20 minutes. In some embodiments, 50% of surface CD22 is internalized within 30 minutes.

[0156] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein have a concentration of about 0.1×10 -6 pg / s / cell, approximately 0.2×10 -6 pg / s / cell, approximately 0.5×10 -6 pg / s / cell, approximately 0.8×10 -6 pg / s / cell, approximately 1.0×10 -6 pg / s / cell, approximately 2.0×10 -6 pg / s / cell, approximately 4.0×10 -6 , about 5.0×10 -6 pg / s / cell, approximately 6.0×10 -6 pg / s / cell, approximately 7.0×10 -6 pg / s / cell, approximately 8.0×10 -6 pg / s / cell, approximately 1.0×10 -5 pg / s / cell, approximately 2.0×10 -5 pg / s / cell, approximately 5.0×10 -5 pg / s / cell or approximately 8.0 × 10 -5 The internalization of Aβ can be induced at a rate of about 0.2×10 -6 In some embodiments, the rate is about 0.5×10 -6 In some embodiments, the rate is about 0.8×10 -6 In some embodiments, the rate is about 1.0×10 -6 In some embodiments, the rate is about 2.0×10 -6 In some embodiments, the rate is about 4.0×10 -6 In some embodiments, the rate is about 6.0×10 -6 In some embodiments, the rate is about 1.0×10 -5 In some embodiments, the rate is about 5.0×10 -5 In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein are at about 0.1-50×10 pg / s / cell. -6 pg / s / cell, approximately 0.1~20×10-6 pg / s / cell, approximately 0.1~10×10 -6 pg / s / cell, approximately 0.5~50×10 -6 pg / s / cell, approximately 0.5~20×10 -6 pg / s / cell, approximately 0.5~10×10 -6 pg / s / cell, approximately 1~50×10 -6 pg / s / cell, approximately 1~20×10 -6 pg / s / cell, approximately 1~10×10 -6 pg / s / cell or approximately 1–6 × 10 -6 The internalization of Aβ can be induced at a rate of about 0.1-50×10 -6 In some embodiments, the rate is about 0.5-50×10 -6 In some embodiments, the rate is about 0.5-10×10 -6 In some embodiments, the rate is about 1-50×10 -6 In some embodiments, the rate is about 1-10×10 pg / s / cell. In some embodiments, the rate is about 1-10×10 -6 In some embodiments, the rate is about 1-6×10 pg / s / cell. In some embodiments, the rate is about 1-6×10 -6 pg / s / cell.

[0157] In some embodiments, the anti-CD22 antibodies or antigen-binding fragments provided herein may be derivatized or linked to another functional molecule (e.g., another peptide or protein) and used in the methods disclosed herein. Thus, in some embodiments, the antibodies and antigen-binding fragments used in the methods disclosed herein include derivatized and otherwise modified forms of the human anti-CD22 antibodies described herein, including immunoadhesion molecules. For example, the antibodies and antigen-binding fragments may 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 (e.g., bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antigen-binding fragment with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0158] One type of derivatized antibody is produced by crosslinking two or more antibodies (e.g., of the same type or of different types to create bispecific antibodies). Suitable crosslinkers include heterobifunctional crosslinkers (e.g., m-maleimidobenzoyl-N-huydroxysuccinimide ester), which have two separately reactive groups separated by a suitable spacer, or homobifunctional crosslinkers (e.g., disuccinimidyle suberate). Such linkers are available from Thermo Scientific, Waltham, Mass.

[0159] In some embodiments, the anti-CD22 antibody or antigen-binding fragment is conjugated to a cytotoxic agent or cytotoxic moiety. In some embodiments, the anti-CD22 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, adriamycin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), 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 tubulysin. In some embodiments, the cytotoxic moiety is an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the trichothecenes. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a trichothene, and CC1065.

[0160] In some embodiments, the anti-CD22 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 include enzymes, such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavin(s); fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), 5-dimethylamine-1-napthalenesulfonyl 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 Ru, 35 S, 47 Sc, 75Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Positron emitting metals; and magnetic metal ions Positron emitting metals; and magnetic metal ions may also be included, but are not limited to:

[0161] The anti-CD22 antibody or antigen-binding fragment described herein can be bound 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, the immobilized anti-CD22 antibody or antigen-binding fragment is used in immunoassay. In some embodiments, the immobilized anti-CD22 antibody or antigen-binding fragment is used in the purification of target antigen (e.g., human CD22). 5.2.2 Further CD22 Antibodies

[0162] Disclosed herein are methods of promoting the removal of Aβ plaques, reducing neuroinflammation, preventing synaptic phagocytosis and / or neuronal death, treating Aβ-related diseases or disorders, and treating diseases or disorders associated with neuroinflammation, using a therapeutically effective amount of an anti-CD22 (e.g., human CD22) antibody or antigen-binding fragment disclosed herein, e.g., SM03, SM06, or a variant or equivalent thereof. Without wishing to be bound by theory, it is understood that SM03, SM06, and other anti-CD22 antibodies disclosed herein can be used to achieve the above-mentioned therapeutic effects because they have at least one of the following functions or activities: (1) bind to an epitope that does not sterically interfere with the interaction of CD22 with Aβ; (2) bind to domain 2 of human CD22; (3) bind to the sequence 161 CLLNFSCYGYPIQ 173(SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 (4) disrupt cis-binding of CD22 homo-clusters; (5) promote cis-trans conversion of 2,6-sialic acid binding of CD22; (6) promote trans-binding of CD22 to neuronal 2,6-sialic acid; (7) promote CD22 internalization, e.g., on microglial cells, Ramos cells and / or B cells, optionally at a rate where 50% of surface CD22 is internalized within 10 minutes; (8) promote internalization of Aβ in microglial cells; (9) suppress pro-inflammatory cytokines, e.g., NFκB signaling and IL-6 secretion; and (10) reduce neuroinflammation.

[0163] Domain 2 and / or sequence of human CD22 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 It is understood that an antibody or antigen-binding fragment that specifically binds to a conformational epitope of human CD22, including (SEQ ID NO: 18), can promote the cis-trans conversion of neuronal 2,6-sialic acid bonds of CD22, thereby inducing rapid internalization of CD22 on microglial cells. Rapid internalization of CD22 then leads to rapid internalization of both CD22-bound Aβ and soluble Aβ by microglial cells without engaging FcγR on the cell surface, thereby avoiding ARIA-E and ARIA-H resulting from FcγR engagement. Furthermore, both internalization of CD22 and removal of Aβ can suppress inflammatory cytokines, such as NFκB signaling and IL-6 secretion, thereby reducing neuroinflammation.

[0164] Thus, methods are also provided herein for selecting for an antibody or antigen-binding fragment that specifically binds CD22 (e.g., human CD22) for at least one of the following functions or activities, and the selected anti-CD22 antibody or antigen-binding fragment may be used in a method of promoting the removal of Aβ plaques, a method of reducing neuroinflammation, a method of preventing synaptic phagocytosis and / or neuronal death, a method of treating an Aβ-related disease or disorder, and / or a method of treating a disease or disorder associated with neuroinflammation: (1) binds to an epitope that does not sterically interfere with the interaction of CD22 with Aβ; (2) binds to domain 2 of human CD22; (3) binds to the sequence 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219 (4) disrupting cis-binding of CD22 homo-clusters; (5) promoting cis-trans conversion of 2,6-sialic acid binding of CD22; (6) promoting trans-binding of CD22 to neuronal 2,6-sialic acid; (7) optionally promoting CD22 internalization on, for example, microglial cells, Ramos cells and / or B cells at a rate such that 50% of CD22 is internalized by 10 minutes; (8) optionally promoting 5.86×10 -6(9) promote Aβ internalization in microglial cells at a rate of pg / s / cell; (10) reduce neuroinflammation; and (11) suppress proinflammatory cytokines, e.g., NFκB signaling and IL-6 secretion. In some embodiments, the selected antibody or antigen-binding fragment may be used in a method of promoting the removal of Aβ plaques. In some embodiments, the selected antibody or antigen-binding fragment may be used in a method of reducing neuroinflammation. In some embodiments, the selected antibody or antigen-binding fragment may be used in a method of preventing synaptic phagocytosis and / or neuronal death. In some embodiments, the selected antibody or antigen-binding fragment may be used in a method of treating an Aβ-related disease or disorder. In some embodiments, the selected antibody or antigen-binding fragment may be used in a method of treating a disease or disorder associated with neuroinflammation.

[0165] For illustrative purposes, epratuzumab (Emab) has been identified as capable of inducing CD22 internalization and has also been confirmed to promote Aβ clearance in microglial cells. Thus, Emab and its functional variants can be used in the methods disclosed herein, including methods of promoting Aβ plaque clearance, reducing neuroinflammation, preventing synaptic phagocytosis and / or neuronal death, treating Aβ-related diseases or disorders, and / or treating diseases or disorders associated with neuroinflammation.

[0166] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is selected from the group consisting of domain 2 of human CD22 or the sequence 161 CLLNFSCYGYPIQ 173 (SEQ ID NO: 17) and 198 VFTRSELKFSPQWSHHGKIVTC 219(SEQ ID NO: 18). In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is selected for its binding to LRID (e.g., U.S. Patent No. 9,371,396 B2).

[0167] The inventors of the present disclosure have found that among different anti-human CD22 antibodies, the closer the binding epitope is to the cell membrane, the slower the internalization rate that the antibody induces.Therefore, by epitope-based selection, i.e., by selecting for anti-CD22 antibodies that bind to epitopes that are far from the cell membrane, the antibody that induces rapid internalization can be identified.Any method of epitope mapping disclosed herein or otherwise known in the art can be used, such as, but not limited to, shotgun mutagenesis, site-directed mutagenesis and alanine scanning; domain or fragment scanning; mutagenesis including, but not limited to, peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display and ribosome / mRNA display); methods involving proteolysis and mass spectroscopy; and structure determination (e.g., X-ray crystallography and NMR).

[0168] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to a discontinuous conformational epitope in domain 2 and induces rapid internalization as disclosed herein may be isolated by screening a recombinant combinatorial antibody library, preferably an scFv phage display library, prepared using human VL and VH cDNA prepared from mRNA derived from human lymphocytes. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available kits for generating phage display libraries (e.g., GE Healthcare Life Sciences Recombinant Antibody Phage System (RAPS); and New England Biolab Ph.D™ Phage Display Library Kit, catalog number E8100S), examples of methods and reagents particularly suitable for use in generating and screening antibody display libraries can be found, for example, in Ladner et al., U.S. Pat. No. 5,223,409; 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.

[0169] In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is greater than or equal to 0.8×10 9 M -1 In some embodiments, the anti-CD22 antibody or antigen-binding fragment is further selected for binding to human CD22 with a K of about 1.2 nM or lower. D In some embodiments, the anti-CD22 antibodies or antigen-binding fragments used in the methods disclosed herein are selected for binding to human CD22 at a concentration of 0.07 RU s -1 or less than 0.015RU s -1 The binding parameters referred to herein may be determined by any method disclosed herein or known in the art, such as surface plasmon resonance. In some embodiments, the anti-CD22 antibody or antigen-binding fragment used in the methods disclosed herein is further selected for its ability to induce CMC activity against surrogate target cells expressing a surface-binding portion of the LRID with an EC50 of 0.2 μg / ml or less.

[0170] In some embodiments, to isolate mouse, recombinant or human antibodies with high affinity and low off-rate constants for human CD22, a parent mouse or chimeric anti-CD22 antibody with high affinity and low off-rate constants for human CD22 (e.g., a hybridoma for a mouse anti-CD22 antibody deposited with the ATCC under Accession No.: 7621) is first used to select human heavy and light chain sequences with similar binding activity for human CD22 using the epitope imprinting method described in PCT Publication No. WO93 / 06213. The antibody library used in this method is preferably an scFv library prepared and screened as described in PCT Publication No. WO92 / 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 as antigen recombinant human CD22 (domains 2-4) and / or an anti-idiotypic antibody specific for the antigen binding site (ABS) of an anti-CD22 antibody (e.g., the LRID described in U.S. Pat. No. 9,371,396B).

[0171] Once the initial human VL and VH segments are selected, a "mix and match" experiment is performed in which different pairs of the initially selected VL and VH segments are screened for human CD22 binding to select a preferred VL / VH pair combination. The affinity and / or specificity of the binding of an antibody to a particular antigen can be increased using the method of "directed evolution" as described in Wu et al., J. Mol. Biol., 294:151 (1999), the contents of which are incorporated herein by reference in their entirety. Additionally, to further improve affinity and / or reduce the off-rate constant for human CD22 (or recombinant CD22(2-4) domain) binding, the VL and VH segments of the preferred VL / VH pair(s) 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 affinity maturation of antibodies during natural immune responses. This in vitro affinity maturation can be accomplished by amplifying the VH and VL regions using PCR primers complementary to the VH or VL CDR3, respectively, which are "spiked" with a random mixture of four nucleotide bases at certain positions, such that the resulting PCR products encode VH and VL segments with random mutations introduced into the VH and / or VL CDR3 regions. These randomly mutated VH and VL segments can be rescreened for binding to human CD22, preferably a recombinant protein containing domains 2-4 of CD22, and / or binding to an anti-idiotypic antibody (e.g., LRID) of an anti-CD22 antibody; sequences exhibiting high affinity and low off-rates for binding to human CD22, preferably recombinant CD22(2-4) domains, and / or anti-idiotypic antibody (e.g., LRID) binding to CD22 can be selected.

[0172] In addition to phage display, the candidate monoclonal antibodies for screening can also be prepared using hybridoma methods well known to those skilled in the art.For example, using hybridoma methods, mice, rats, rabbits, hamsters or other suitable host animals are immunized as described above.In some embodiments, lymphocytes are immunized in vitro.In some embodiments, the immunization antigen is a human protein or a fragment thereof.In some embodiments, the immunization antigen is a human protein or a fragment thereof.

[0173] After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol. Hybridoma cells are selected using specialized media known in the art, and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas producing monoclonal antibodies against 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 antibodies of the desired specificity, affinity and / or activity are identified, clones can be subcloned by limiting dilution or other techniques. Hybridomas can be grown in in vitro culture using standard methods, or in vivo in animals as ascites tumors. Monoclonal antibodies can be purified from culture medium or ascites fluid according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis and dialysis.

[0174] Further methods of screening for antibodies or antigen-binding fragments that disrupt CD22 binding in cis have been previously disclosed by the inventors of the present disclosure (e.g., WO2020078453A1, which is incorporated herein by reference in its entirety). In some embodiments, the screening method includes using an engineered cell line expressing a fusion protein comprising a non-internalizing human CD22 domain 1-7 and a domain containing a 2,6-sialic acid ligand by comparing the binding of an exogenous probe containing multiple 2,6-sialic acids to the engineered cell line in the presence and absence of the antibody, and an antibody is identified as capable of disrupting CD22 cis-binding if it restores binding of the exogenous probe to the engineered cell line.

[0175] In some embodiments, the fusion protein comprises human CD22 domains 1-7 and glycophorin A. In some embodiments, the fusion protein comprises the extracellular domain of human CD22 fused to the transmembrane and cytoplasmic portions of glycophorin A. In some embodiments, the fusion protein comprises the extracellular domain of human CD22 fused to the glycophosphatidylinositol signal sequence isolated from the decay accelerating factor (DAF) protein.

[0176] In some embodiments, the fusion protein has an amino acid sequence that comprises at least 95% identical to SEQ ID NO: 15. In another embodiment, the fusion protein has an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15. In another embodiment, the fusion protein has the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion protein has an amino acid sequence that comprises at least 95% identical to SEQ ID NO: 16. In another embodiment, the fusion protein has an amino acid sequence that is at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 15. In another embodiment, the fusion protein has the amino acid sequence of SEQ ID NO: 16.

[0177] In some embodiments, the exogenous probe is a biotin-conjugated polyacrylamide substituted with α2-6-sialyllactose (6′PAA-B; Glycotech).

[0178] In some embodiments, non-human antibodies identified as having a therapeutic use as disclosed herein through the screening methods disclosed herein may be humanized, where certain sequences or regions of the antibody are modified to increase similarity to antibodies naturally produced in humans, hi some embodiments, the antigen-binding domain portion is humanized.

[0179] Often, framework residues in framework regions can be replaced with corresponding residues from CDR donor antibodies to alter, preferably improve, antigen binding. These framework replacements are identified by methods well known in the art, for example, by modeling the interaction of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, for example, Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

[0180] A humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Thus, a humanized antibody contains one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Methods for antibody humanization, including CDR-grafting, are well known in the art (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 No. 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 6,548,640, each of which is incorporated herein by reference in its entirety). In such humanized antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. 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 include veneering or resurfacing (see, e.g., European Patent Nos. 592,106 and 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), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety), as well as techniques such as those described in, e.g., U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO9317105, 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 JS, Gene, 150(2):409-10 (1994) and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994).

[0181] The human variable domains of both light and heavy chains used in making humanized antibodies are selected to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of rodent antibodies is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (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 derived from the consensus sequence of all human antibodies of 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 by reference in their entireties.

[0182] Antibodies can be humanized with high affinity for target antigens and retention of other favorable biological properties. For example, humanized antibodies can be prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible role of the residues in the functionality of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences, so that the desired antibody characteristics, such as increased affinity for the target antigen, are achieved. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0183] After screening and isolation of the anti-CD22 antibodies disclosed herein from a 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 desired, the nucleic acid can be further manipulated (e.g., linked to nucleic acids encoding additional immunoglobulin domains, e.g., additional constant regions) to create other antibody forms disclosed herein or known in the art. Methods for expressing recombinant human antibodies isolated by screening combinatorial libraries and introduced into mammalian host cells, including cloning the DNA encoding the antibody into a recombinant expression vector, are well known in the art.

[0184] In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for (1) binding to an epitope that does not sterically interfere with the interaction of CD22 with Aβ, and (2) disrupting the cis-binding of CD22 homo-clusters, promoting cis-trans conversion of 2,6-sialic acid binding of CD22, and / or promoting trans-binding of CD22 to neuronal 2,6-sialic acid. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is directly selected for its function of promoting CD22 internalization, promoting Aβ internalization in microglial cells, preferably without engaging FcγR on the cell surface, reducing neuroinflammation, inhibiting NFκB signaling in microglial cells, inhibiting IL-6 secretion by microglial cells, or any combination thereof.

[0185] In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of promoting CD22 internalization. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of promoting Aβ internalization in microglial cells, preferably without engaging FcγR on the cell surface. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of reducing neuroinflammation. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of suppressing NFκB signaling in microglial cells. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of suppressing IL-6 secretion by microglial cells. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of suppressing neuroinflammation. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of suppressing synaptic phagocytosis. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is selected for its function of suppressing neuronal death.

[0186] The anti-CD22 antibodies or antigen-binding fragments described herein can be tested for binding to human CD22, for example, by standard ELISA. Briefly, microtiter plates are coated with purified CD22 and then blocked with bovine serum albumin. Dilutions of antibody (e.g., dilutions of plasma from mice immunized with CD22) are added to each well and incubated. The plates are washed and incubated with a secondary reagent conjugated to horseradish peroxidase (HRP) (e.g., for human antibodies, goat anti-human IgG Fc-specific polyclonal reagent). After washing, the plates can be developed and analyzed by spectrophotometer. Sera from immunized mice can then be further screened by flow cytometry for binding to cell lines expressing human CD22, but not to control cell lines that do not express CD22. Briefly, binding of anti-CD22 antibodies can be evaluated by incubating CD22-expressing CHO cells with anti-CD22 antibodies. The cells can be washed and binding can be detected with an anti-human IgG Ab. Flow cytometric analysis can be performed using a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.). Mice exhibiting the highest titers can be used for fusions.

[0187] The above ELISA assay can be used to screen for antibodies and thus hybridomas that produce antibodies that show positive reactivity with CD22 immunogen. Hybridomas that produce antibodies that bind to CD22 with high affinity can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parent cell (by ELISA) can then be selected for making a cell bank and for antibody purification.

[0188] To purify anti-CD22 antibodies, selected hybridomas can be grown for monoclonal antibody purification. The supernatant can be filtered and concentrated prior to affinity chromatography. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged and the concentration can be determined. The monoclonal antibody can be aliquoted and stored.

[0189] Various methods and assays for determining the structural and functional properties of antibodies and antigen-binding fragments are known in the art.For example, to determine whether selected anti-CD22 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL).Biotinylated MAb binding can be detected using streptavidin-labeled probes.Competition studies using unlabeled and biotinylated monoclonal antibodies can be carried out using CD22-coated ELISA plates.

[0190] Flow cytometry can be used to test the binding of monoclonal antibodies to live cells expressing CD22. Briefly, CD22-expressing cell lines (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA for 1 hour at 4°C. After washing, cells are reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as primary antibody staining. Samples can be analyzed by a FACScan instrument that uses light and side scatter properties to gate on single cells, and binding of the labeled antibody is determined. An alternative assay using a fluorescent microscope can be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescent microscopy. This method allows visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

[0191] Methods for analyzing the binding affinity, cross-reactivity and binding kinetics of various anti-CD22 antibodies include standard assays known in the art, such as biolayer interferometry (BLI) using a Gator system (Probe Life) or an Octet-96 system (Sartorius AG), or BIACORE™ surface plasmon resonance (SPR) analysis using a BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0192] The assay for testing or screening anti-CD22 antibody or antigen-binding fragment for the functional properties disclosed above is well known in the art.Any method disclosed herein or otherwise known in the art can be used in the screening method disclosed herein.For example, the functional assay that can be used in the screening method disclosed herein includes luciferase assay for measuring NFκB signaling, ELISA assay for measuring IL-6 release, ELISA or Western blot assay for measuring synaptic phagocytosis, and MTT assay for measuring neuronal death, all of which are disclosed in the following experimental section.

[0193] Also provided herein, in some embodiments, are anti-CD22 antibodies and antigen-binding fragments identified or produced using the methods described herein, and their therapeutic uses. 5.3 Method of production

[0194] Anti-CD22 antibodies and antigen-binding fragments thereof that may be used in the methods disclosed herein, including but not limited to monoclonal antibodies, chimeric antibodies, human antibodies and humanized antibodies, may be prepared by any method disclosed herein or otherwise known in the art.

[0195] Methods for producing antibodies are well known in the art. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563, 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety.

[0196] In some embodiments, the antibodies or antigen-binding fragments that can be used 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, for example, by introducing a recombinant expression vector into a host cell, a recombinant combinatorial human antibody library, an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (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 into other DNA sequences.

[0197] In some embodiments, antibodies and antigen-binding fragments can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To recombinantly express antibodies, one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of antibodies are introduced into host cells, so that the light and heavy chains are expressed in host cells and preferably secreted into the medium in which the host cells are cultured, and the antibody can be recovered from the medium. Standard recombinant DNA methodologies, such as those described in 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 Assoviates, (1989) and U.S. Patent No. 4,816,397, are used to obtain antibody heavy and light chain genes, to incorporate these genes into recombinant expression vectors, and to introduce the vectors into host cells.

[0198] To express a recombinant antibody or antigen fragment, such as SM03, SM06, or SM03 / SM06-related antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of hybridomas for mouse antibody light and heavy chain variable sequences using polymerase chain reaction (PCR), or by oligo synthesis based on the encoded amino acid sequences of the designed light and heavy chain variable sequences, using standard methods known to those skilled in the art. The coding DNA sequences can be further optimized to facilitate mammalian expression of the obtained antibodies.

[0199] Once the VH and VL fragments are obtained for the mouse antibody, these sequences can be mutated to encode a framework patched version of SM03 (i.e., SM06), methods of which are described in Chinese Patent Nos. 01144894.6 and 03123054.7 and U.S. Patent Nos. 7,321,026 B2 and 7,338,659 B2, both of which are incorporated herein by reference in their entireties.

[0200] Once DNA fragments encoding SM03 or SM06 or SM03 / SM06-related VH and VL segments are obtained (e.g., by amplification and mutagenesis of the original mouse VH and VL genes as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, into Fab fragment genes, or into scFv genes. In these manipulations, the VL-encoding DNA fragment or the VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, for example, an antibody constant region or a flexible linker. The term "operably linked" as used in this context is intended to mean that the two DNA fragments are connected such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0201] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, 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 encompassing 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 is most preferably an IgG1 or IgG4 constant region. For a 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.

[0202] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, 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 encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is most preferably a kappa constant region.

[0203] To prepare an scFv gene, a DNA fragment encoding a VH and a DNA fragment encoding a VL are operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions connected by the 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).

[0204] To express the antibody or antigen-binding fragment that can be used in the methods disclosed herein, the DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector, so that the genes are operably linked to transcription and translation control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcription and translation control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequences 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, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites exist). In some embodiments, prior to insertion of SM03, SM06 or SM03 / SM06-related light or heavy chain sequences, the expression vector already carries antibody constant region sequences. For example, one approach to convert SM03 or SM06 or SM03 / SM06-related VH and VL sequences into full-length antibody genes is to insert them into an expression vector already encoding heavy and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) in the vector and the VL segment is operably linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0205] In addition to the antibody chain genes, the recombinant expression vectors provided herein may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. 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. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990). As one of ordinary skill in the art will appreciate, the design of the expression vector, including the selection of regulatory sequences, depends on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from immunoglobulin heavy chain (IgH) enhancer (Gillies et al. (1983) Cell 33:717-728), metallothionein (MT), cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Pat. Nos. 5,665,578; 5,168,062; 4,510,245; and 4,968,615.

[0206] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors provided herein may carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells in conjunction with methotrexate selection / amplification), the glutamate synthase (GS) gene and the neo gene (for G418 selection).

[0207] For the expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, protoplast fusion, etc. Although antibodies and antigen-binding fragments can be produced in either prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, particularly mammalian host cells, is preferred, since such host cells are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies.

[0208] Preferred mammalian host cells for expressing recombinant antibodies used in the methods described herein include SP2 / 0 myeloma cells, NSO myeloma cells, COS cells and Chinese hamster ovary (CHO) cells (e.g., dfhr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4200, used with the DHFR selectable marker described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621). When an expression vector encoding a recombinant antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0209] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. Variations of the above procedures are expressly contemplated herein. For example, it may be desirable to transfect host cells with DNA encoding either the light or heavy chains (but not both) of the antibodies used in the methods disclosed herein. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to CD22. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies provided herein. Furthermore, bifunctional antibodies, in which one heavy chain and one light chain are antibodies that specifically bind to human CD22 and the other heavy and light chains are specific for an antigen other than CD22, can be produced by crosslinking the antibody of the present invention to a second antibody by standard chemical crosslinking methods.

[0210] In some embodiments of a system for recombinant expression of an antibody or antigen-binding fragment thereof that can be used in the methods disclosed herein, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into SP2 / 0 cells by electroporation. In some embodiments of the expression system, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into CHO cells by standard techniques such as lipofection.

[0211] Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to mouse or human immunoglobulin heavy chain (IgH), CMV enhancer, metallothionein or AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, allowing for selection of SP2 / 0 cells transfected with the vector using methotrexate selection / amplification. Alternatively, a recombinant expression vector containing antibody heavy and light chain genes operably linked to mouse or human IgH, CMV enhancer / AdMLP / metallothionein promoter regulatory elements and a DHFR gene can be used to transfect SP2 / 0 or CHO cells that are dhfr-. SP2 / 0 or CHO cells transfected with the vector can be selected, and the level of gene expression in the vector can be amplified by increasing the level of methotrexate in the culture. The selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibodies are recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. 5.4 Pharmaceutical Compositions

[0212] Also provided herein are pharmaceutical compositions comprising anti-CD22 antibodies or antigen-binding fragments (e.g., SM03, SM06) that can be used in the methods disclosed herein. In some embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of anti-CD22 antibodies or antigen-binding fragments disclosed herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are useful in treating Aβ-related diseases or disorders or neuroinflammation-related diseases or disorders. In some embodiments, the pharmaceutical compositions are useful in treating AD. In some embodiments, the pharmaceutical compositions are useful in inhibiting AD progression in a subject (e.g., a human patient). In some embodiments, the pharmaceutical compositions are useful in ameliorating cognitive impairment in a subject (e.g., a human patient).

[0213] The amount of therapeutic antibody that can be combined with carrier material in the pharmaceutical composition disclosed herein can vary. In some embodiments, the amount of antibody present in the pharmaceutical composition is an amount that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient combined with a pharma- ceutically acceptable carrier.

[0214] The pharmaceutical compositions provided herein include an anti-CD22 antibody or antigen-binding fragment provided herein, e.g., SM03, SM06, or SM03 / SM06 related antibodies, or an anti-CD22 antibody or antigen-binding fragment identified by the methods disclosed herein. The anti-CD22 antibody or antigen-binding fragment may be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein include a soluble anti-CD22 antibody or antigen-binding fragment provided herein at 1-1000 mg / ml. In some embodiments, the pharmaceutical compositions include a soluble anti-CD22 antibody or antigen-binding fragment provided herein at 10-500 mg / ml, 10-400 mg / ml, 10-300 mg / ml, 10-200 mg / ml, 10-100 mg / ml, 20-100 mg / ml, or 50-100 mg / ml. In some embodiments, the pharmaceutical compositions provided herein comprise an anti-CD22 antibody or antigen-binding fragment provided herein at about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 120 mg / ml, about 150 mg / ml, about 180 mg / ml, about 200 mg / ml, about 300 mg / ml, about 500 mg / ml, about 800 mg / ml, or about 1000 mg / ml. Dosage can be readily adjusted by one of skill in the art; for example, a decrease in purity will require an increase in dosage.

[0215] The pharmaceutical compositions provided herein can be in various forms.These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration and therapeutic application.The examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

[0216] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the pharmaceutical compositions provided herein are in the form of an injectable or infusible solution. In some embodiments, the pharmaceutical compositions are aqueous formulations. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation that contains at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution that contains at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension that contains at least 50% w / w water. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration.

[0217] In some embodiments, the pharmaceutical compositions disclosed herein are freeze-dried, to which the physician or the patient adds solvents and / or diluents prior to use.

[0218] The pharmaceutical compositions provided herein may include pharmaceutically acceptable carriers.Pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Examples include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof.In some embodiments, pharmaceutically acceptable carriers include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride.

[0219] In some embodiments, the pharma- ceutically acceptable carrier further comprises minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antigen-binding fragment. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., anti-CD22 antibody or antigen-binding fragment) may be coated with a material to protect the active ingredient from the action of acids and other natural conditions that may inactivate the active ingredient.

[0220] Also provided herein are kits for the preparation of pharmaceutical compositions having an anti-CD22 antibody or antigen-binding fragment disclosed herein, e.g., SM03, SM06, or SM03 / SM06-related antibody. In some embodiments, the kit comprises an anti-CD22 antibody or antigen-binding fragment disclosed herein and a pharma- ceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise an anti-CD22 antibody or antigen-binding fragment disclosed herein for administration to a subject. In a specific embodiment, the kit comprises instructions for preparation and / or administration of the anti-CD22 antibody or antigen-binding fragment.

[0221] In some embodiments, the pharmaceutical composition or formulation disclosed herein comprises: (a) an anti-CD22 antibody or antigen-binding fragment disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a bulking agent; and / or (f) a surfactant. In some embodiments, the pharmaceutical composition or formulation is stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years, or longer. In some embodiments, the pharmaceutical composition or formulation is stable when stored at 4°C, 25°C, or 40°C. In some embodiments, pharmaceutical compositions or formulations that improve the stability of anti-CD22 antibodies or antigen-binding fragments to allow for their long-term storage are also provided herein. The pharmaceutical compositions disclosed herein may further comprise one or more of a preservative, an isotonicity agent, a chelating agent, a stabilizer, and / or a surfactant, as well as various combinations thereof. The use of preservatives, isotonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to Remington: The Science And Practice Of Pharmacy, 19th edition, 1995.

[0222] Buffers useful in the pharmaceutical compositions or formulations disclosed herein can be weak acids or weak bases used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity and other properties can also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate and phosphate. In some embodiments, the buffer includes histidine (e.g., L-histidine), along with an isotonicity agent and potentially pH adjustment with an acid or base as known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10 or between about 4 and about 8.

[0223] Stabilizers are added to pharmaceutical products to stabilize the product. Such agents can stabilize proteins in different ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine or threonine, carbohydrates such as glucose, sucrose, trehalose, rafftnose or maltose, polyols of any type and molecular weight such as glycerol, mannitol, sorbitol, cyclodextrin or dextran, or PEG. In some embodiments, the stabilizer is selected to maximize the stability of FIX polypeptide in lyophilized preparations. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0224] Bulking agents may be added to a pharmaceutical composition or formulation to add volume and mass to the product, thereby facilitating its accurate measurement and handling. Common bulking agents include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0225] Surfactants are amphiphilic substances that have lyophilic and lyophobic groups.Surfactants can be anionic, cationic, zwitterionic or nonionic.Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxides, polypropylene oxides, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyl dimethylamine oxide.In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0226] The pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable antioxidants.The examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0227] These compositions also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0228] Pharmaceutical compositions or preparations must typically be sterile and stable under the conditions of manufacture and storage. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Sterile injectable solutions can be prepared by incorporating therapeutic antibodies or antigen-binding fragments in the required amount in a suitable solvent with one or a combination of the components listed above, and then optionally sterilizing by filtration. The use of such media and agents for pharmaceutically active substances is known in the art. In general, dispersions are prepared by incorporating active compounds into a sterile vehicle that contains a basic dispersion medium and the required other components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization), which produce a powder of active ingredient plus any additional desired ingredients from its solution that has previously been sterile-filtered.

[0229] The pharmaceutical compositions disclosed herein can be prepared with carriers that protect active ingredient against rapid release, such as sustained release formulations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid.Many methods for preparing such formulations are patented or generally known to those skilled in the art.See, for example, Sustained And Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0230] In some embodiments, the anti-CD22 antibody or antigen-binding fragment described herein can be formulated to ensure proper distribution in vivo. For example, the BBB excludes many highly hydrophilic compounds. To facilitate the therapeutic antibodies described herein to cross the BBB, they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.), mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273. 5.5 Treatment Method

[0231] As described in the above section, medical uses of anti-CD22 antibodies and antigen-binding fragments (e.g., SM03 or SM06) in treating Aβ or neuroinflammation-related diseases or disorders are provided herein. Any anti-CD22 antibody or antigen-binding fragment disclosed herein or identified using the screening method disclosed herein can be used in the methods disclosed herein. In some embodiments, the therapeutic antibody is SM03. In some embodiments, the therapeutic antibody is SM06. In some embodiments, methods of promoting Aβ plaque removal in a subject in need thereof are provided herein. In some embodiments, methods of treating Aβ-related diseases or disorders in a subject in need thereof are also provided herein. In some embodiments, methods of reducing neuroinflammation in a subject in need thereof are also provided herein. In some embodiments, methods of treating neuroinflammation-related diseases or disorders in a subject in need thereof are also provided herein. In some embodiments, methods of treating neuroinflammation-related diseases or disorders in a subject in need thereof are also provided herein. In some embodiments, methods provided herein prevent synaptic phagocytosis and neuronal death. In some embodiments, the methods provided herein prevent synaptic phagocytosis and neuronal death by at least 20%, at least 30%, at least 40%, at least 50% or at least 60%. In some embodiments, the subject is a human. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human CD22.

[0232] The methods of promoting Aβ plaque removal, reducing neuroinflammation, treating an Aβ-related disease or disorder, and / or treating a disease or disorder disclosed herein include administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to CD22, wherein the antibody or antigen-binding fragment (a) promotes cis-to-trans conversion of CD22 and / or (b) induces internalization of CD22.

[0233] In some embodiments, the Aβ-related disease or disorder and / or neuroinflammatory-related disease or disorder can be clinical or preclinical AD, prodromal AD, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia, cognitive impairment, memory loss, or vascular disorders caused by pathogenic Aβ peptides in the blood vessels.

[0234] Suitable subjects for the method of the present invention include human patients for whom removal of Aβ plaques and / or reduction in neuroinflammation is desired. In some embodiments, the subject treated with the method disclosed herein is diagnosed with an Aβ-related or neuroinflammatory-related disease or disorder, which may be clinical or preclinical AD, prodromal AD, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disorder caused by pathogenic Aβ peptides in blood vessels. In some embodiments, the subject treated with the method disclosed herein is at risk of developing an Aβ-related or neuroinflammatory-related disease or disorder, which may be clinical or preclinical AD, prodromal AD, Down's syndrome, clinical or preclinical amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, preeclampsia, cognitive impairment, memory loss, or vascular disorder caused by pathogenic Aβ peptides in blood vessels. The subject can be a mammal, hi some embodiments, the subject is a human.

[0235] In some embodiments, the subject is diagnosed with AD. In some embodiments, the subject is at risk of developing AD. In some embodiments, the subject has preclinical AD. In some embodiments, the subject has clinical AD. In some embodiments, the subject has prodromal AD. In some embodiments, the subject treated with the methods disclosed herein has been treated with standard therapy for AD. In some embodiments, the subject has not been previously treated.

[0236] The anti-CD22 antibodies or antigen-binding fragments (e.g., SM03 or SM06) or pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, including, but not limited to, intravenous, subcutaneous, intramuscular, intracranial, intrathecal, intraventricular, intraperitoneal, spinal, intranasal, intrapleural, topical, or intradermal administration.

[0237] In some embodiments, an anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) or pharmaceutical composition provided herein may be administered to a subject using parenteral administration. The phrase "parenteral administration" as used herein means a mode of administration other than enteral and topical administration, usually by injection, including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, an anti-CD22 antibody or antigen-binding fragment is administered by intravenous infusion or injection. In some embodiments, an anti-CD22 antibody or antigen-binding fragment is administered by intramuscular injection. In some embodiments, an anti-CD22 antibody or antigen-binding fragment is administered by subcutaneous injection.

[0238] The blood-brain barrier tightly regulates the transport of substances in and out of the brain. As known in the art, patients with AD are associated with vascular leakage, and IgG penetration into the brain can reach approximately 0.2%. Thus, the therapeutic antibody delivered systematically can cross the BBB and reach the lesion site. Alternatively, in some embodiments, the antibody or antigen-binding fragment provided herein can be delivered locally using intracranial administration. In some embodiments, intraventricular administration is employed.

[0239] Anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) or pharmaceutical composition provided herein can be administered using medical devices known in the art.For example, in some embodiments, needleless hypodermic injection devices can be used, such as the devices disclosed in U.S. Patent No. 5,399,163; No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,790,824; or No. 4,596,556. Examples of well-known implants and modules for use as described herein include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drug therapy at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicine through the skin; U.S. Patent No. 4,447,233, which discloses a drug therapy infusion pump for delivering drug therapy at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

[0240] In some embodiments, the anti-CD22 antibody or antigen-binding fragment disclosed herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier.The therapeutic antibody can also be enclosed in a hard or soft shell gelatin capsule, compressed into a tablet, or directly incorporated into the subject's diet.For oral therapeutic administration, the therapeutic antibody can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0241] The methods provided herein include administering a therapeutically effective amount of an anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) described herein. The actual dosage level of the therapeutic antibody may be varied to obtain an amount effective to achieve the desired therapeutic response for a particular patient without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including factors well known in the medical arts, such as the activity of the particular composition described herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, relative health and previous medical history of the patient being treated.

[0242] In general, dosages may range from, for example, about 0.1 to 100 mg per kg of host body weight for a single dose. In some embodiments, the anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) is administered at about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 1 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 5 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 10 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 20 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 40 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 60 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at about 100 mg / kg.

[0243] In some embodiments, the anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) is administered at a dose within the range of about 1-5 mg / kg, about 1-10 mg / kg, about 1-20 mg / kg, about 1-50 mg / kg, about 1-100 mg / kg, about 5-10 mg / kg, about 5-20 mg / kg, about 5-50 mg / kg, about 5-100 mg / kg, about 10-50 mg / kg, or about 10-100 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at a dose within the range of about 1-5 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at a dose within the range of about 1-10 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at a dose within the range of about 1-50 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at a dose in the range of about 10-50 mg / kg. In some embodiments, the anti-CD22 antibody or antigen-binding fragment is administered at a dose in the range of about 10-100 mg / kg.

[0244] In some embodiments, the methods provided herein include administering an anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) at a dose of about 10 to 2000 mg. In some embodiments, the dose is about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, or about 2000 mg. In some embodiments, the antibody is administered at a dose of 100 mg. In some embodiments, the antibody is administered at a dose of 300 mg. In some embodiments, the antibody is administered at a dose of 600 mg. In some embodiments, the antibody is administered at a dose of 900 mg. In some embodiments, the antibody is administered at a dose of 1200 mg.

[0245] In some embodiments, the methods provided herein include administering an anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) at a dose in the range of about 10-50 mg, 10-100 mg, 10-200 mg, 100-300 mg, 100-500 mg, 300-600 mg, 300-900 mg, 300-1200 mg, 600-1200 mg, 600-1800 mg, or 1000-2000 mg. In some embodiments, the antibody is administered at a dose in the range of 100-500 mg. In some embodiments, the antibody is administered at a dose in the range of 300-600 mg. In some embodiments, the antibody is administered at a dose in the range of 300-900 mg. In some embodiments, the antibody is administered at a dose in the range of 600-1200 mg.

[0246] During treatment, it is common to start with a lower dose, which is then increased to a target dose. For illustrative purposes, in some embodiments, the methods provided herein include administering an anti-CD22 antibody or antigen-binding fragment at a dose of about 100 mg, which is gradually increased to a target dose of about 600 mg.

[0247] The subject may be administered such doses once a day, every other day, once a week, once every two weeks, once a month, or according to any other schedule determined by empirical analysis. Exemplary treatments entail administration in multiple dosages over an extended period of time, e.g., a period of at least six months. In some embodiments, the methods provided herein include administering an anti-CD22 antibody or antigen-binding fragment once a week. In some embodiments, the methods include administering once every two weeks. In some embodiments, the methods include administering once a month. In some embodiments, the anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) is administered subcutaneously once a week, once every two weeks, or once a month. In some embodiments, the anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) is administered intravenously once a week, once every two weeks, or once a month.

[0248] The anti-CD22 antibody or antigen-binding fragment (e.g., SM03 or SM06) may be administered for up to 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, or 36 months, as needed and as appropriate. In some embodiments, the treatment lasts for at least 3 months. In some embodiments, the treatment lasts for at least 6 months. In some embodiments, the treatment lasts for at least 12 months. In some embodiments, the treatment lasts for at least 24 months.

[0249] For example, all permutations and combinations of the various embodiments of rounds of administration, dosages, frequency of treatment, and length of treatment are expressly contemplated herein and may be employed in the therapeutic methods disclosed herein.

[0250] For illustrative purposes, the following treatment regimens may be employed in the methods disclosed herein involving administration of an anti-CD22 antibody or antigen-binding fragment disclosed herein (e.g., SM03 or SM06) or identified in the methods disclosed herein.

[0251] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously at a dose of about 10 mg / kg every 4 weeks and at least 21 days apart. In some embodiments, the following titration schedule is included: infusions 1-2: 1 mg / kg IV; infusions 3-4: 3 mg / kg IV; infusions 5-6: 6 mg / kg IV; infusions 7 and beyond: 10 mg / kg IV.

[0252] In some embodiments, the therapeutic antibody is administered intravenously or subcutaneously at a single dose of 10, 20 or 40 mg / kg, a second dose of 10 mg / kg every other week for 24 weeks, and a third dose of 10 or 20 mg / kg every month for 16 months.

[0253] In some embodiments, therapeutic antibodies are administered intravenously or subcutaneously at a dose of about 250 mg once a week or 500 mg once every two weeks for up to two years. In some embodiments, treatment begins with a monthly shot of about 120 mg.

[0254] Dosage regimes can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for the mammalian subject to be treated; each unit contains a predetermined amount of therapeutic antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. It should be noted that suitable dosages vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regime should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0255] In the treatment of Aβ-related diseases or disorders or neuroinflammation-related diseases or disorders, sometimes the disease or disorder may be cured with the methods provided herein, but any clinical improvement constitutes a benefit. In some embodiments, the methods provided herein reduce amyloid by an average of about 50 centiloid (CL), about 60 CL, about 70 CL, about 80 CL, about 90 CL, about 95 CL, or about 99 CL. In some embodiments, the methods provided herein reduce amyloid by an average of about 50 CL. In some embodiments, the methods provided herein reduce amyloid by an average of about 80 CL. In some embodiments, the methods provided herein reduce amyloid by an average of about 90 CL. In some embodiments, the methods provided herein reduce amyloid by an average of 50-100 CL, 60-100 CL, 70-100 CL, 80-100 CL, or 90-100 CL. In some embodiments, the methods provided herein reduce amyloid by an average of 50-100 CL. In some embodiments, the methods provided herein reduce amyloid by an average of 90-100 CL. In some embodiments, the methods provided herein reduce neuroinflammation. In some embodiments, the methods provided herein reduce vasogenic edema. In some embodiments, the methods provided herein prevent synaptic phagocytosis and neuronal death. In some embodiments, the methods provided herein prevent the onset of AD or slow or stop progression AD. In some embodiments, the methods provided herein ameliorate symptoms of AD. In some embodiments, the methods provided herein ameliorate cognitive impairment. In some embodiments, the methods provided herein delay the onset of cognitive impairment.

[0256] In comparison to therapeutic antibodies that cross-link FcγR, the anti-CD22 antibodies disclosed herein have no or reduced vascular side effects (i.e., ARIA-E, ARIA-H). Additionally, in some embodiments, the methods provided herein reduce vasogenic edema.

[0257] The anti-CD22 antibody or antigen-binding fragment disclosed herein can be administered by various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. In some embodiments, the therapeutic antibody can be prepared with a carrier that protects it against rapid release, such as sustained release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyethylene glycol (PEG), polyanhydrides, polyglycolic acid, collagen, polyorthoesteers, and polylactic acid, can be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, for example, Sustained And Controlled Release Drug Delivery Systems, JR Robinson ed., Marcel Dekker, Inc., New York, 1978. In therapeutic applications, relatively high dosages at relatively short intervals are sometimes required until the progression of the disease is reduced or terminated, and until the patient shows partial or complete remission of the symptoms of the disease.

[0258] Combination therapy using agents with different mechanisms of action may produce additive or synergistic effects. Combination therapy may allow for lower doses of each agent than those used in monotherapy, thereby reducing toxic side effects and / or increasing the therapeutic index of the agents disclosed herein. Combination therapy may reduce the likelihood of drug resistance developing. In some embodiments, the additional treatment produces an increase in the therapeutic index of the antibody or antigen-binding fragment or pharmaceutical composition described herein. In some embodiments, the additional treatment produces a decrease in the toxicity and / or side effects of the antibody or antigen-binding fragment or pharmaceutical composition described herein. In some embodiments, the anti-CD22 antibody or antigen-binding fragment or pharmaceutical composition described herein may be administered in combination with an additional treatment.

[0259] In some embodiments, the second therapeutic agent is an anti-Aβ antibody, an anti-CD33 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. In some embodiments, the second therapeutic agent can be a second antibody that suppresses the release of pro-inflammatory cytokines, or an agent that enhances microglial cell phagocytosis activity.

[0260] In some embodiments, the second therapeutic agent is an anti-Aβ antibody. The anti-Aβ antibody may be selected from the group consisting of aducanumab, donanemab, gantenerumab, lecanemab, bapineuzumab and solanezumab. In some embodiments, the second therapeutic agent is aducanumab. In some embodiments, the second therapeutic agent is donanemab. In some embodiments, the second therapeutic agent is gantenerumab.

[0261] In some embodiments, the second therapeutic agent is an anti-CD33 antibody. In some embodiments, the second therapeutic agent is selected from the group consisting of AL003 (AbbVie), gemtuzumab, lintuzumab, ozogamicin, vadastuximab butarilin, and BI836858. In some embodiments, the second therapeutic agent is AL003. In some embodiments, the second therapeutic agent is gemtuzumab. In some embodiments, the second therapeutic agent is lintuzumab.

[0262] In some embodiments, the second therapeutic agent is a tau aggregation inhibitor. In some embodiments, the second therapeutic agent is the methylene blue derivative LMTX (also known as LMTM or TRx0237) or curcumin.

[0263] In some embodiments, the second therapeutic agent is a tau protein modulator. In some embodiments, the second therapeutic agent is memantine, sodium selenate, alvocidib, seliciclib, tideglusib, lithium, salsalate or MK-8719.

[0264] In some embodiments, the second therapeutic agent is a cholinesterase inhibitor or an acetylcholinesterase inhibitor. In some embodiments, the second therapeutic agent is donepezil, rivastigmine or galantamine.

[0265] In some embodiments, the second therapeutic agent is an NMDA antagonist. In some embodiments, the second therapeutic agent is memantine.

[0266] The second therapeutic agent can be administered before, simultaneously, or subsequently to the administration of the anti-CD22 antibody or antigen-binding fragment or pharmaceutical composition described herein.Combined administration can include co-administration in a single pharmaceutical formulation or using separate formulations, or sequential administration in any order, but generally within a period during which all active agents can simultaneously exert their biological activity.Those skilled in the art can easily determine the appropriate regimen for administering the pharmaceutical composition described herein and the additional treatment in combination, including the timing and dosage of the additional agent used in combination therapy, based on the needs of the subject being treated.

[0267] All articles, publications, and patents cited herein are incorporated by reference to the same extent as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not and should not be construed as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0268] Unless the context indicates otherwise, it is specifically contemplated that the various features described herein can be used in any combination.

[0269] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. EXAMPLES

[0270] 5.6 Experiments 5.6.1 Example 1 CD22 expression on microglial cells. Using immunohistochemical procedures, immunocytochemical procedures and database searches, CD22 was found to be expressed on multiple types of cells, including Ramos cells, B cells and microglial cells (e.g., HMC-3 cells and BV-2 cells). [Table 3] Ramos: human Burkitt's lymphoma cells; HMC-3: human microglial cells; BV-2: mouse microglial cells.

[0271] Consistently, we also detected binding of various anti-CD22 antibodies to the above cell lines, including microglial cells (eg, HMC-3 cells and BV-2 cells), as shown in the table below. [Table 4] + indicates positive binding, - indicates no binding. 5.6.2 Example 2 In vitro binding of CD22 to oligomeric Aβ1-42.

[0272] The Octet 96e SPR system was used to analyze the in vitro binding of CD22 to Aβ1-42. A streptavidin biosensor was loaded with 50 μg / mL biotinylated oligomeric Aβ1-42; the interaction with various concentrations of CD22 was detected using the biosensor. The steps were as follows: first, the streptavidin biosensor was loaded in 50 μg / mL biotinylated oligomeric Aβ1-42 for 4 minutes; second, the biosensor was washed with kinetic buffer (PBS + 0.02% Tween® 20, 0.1% BSA) for 10 seconds; third, the biosensor was loaded in various concentrations of the extracellular domain of CD22 (Peprotech) for 2 minutes for association. As shown in Figure 1, CD22 has a K of approximately 2.79 nM. D It bound to oligomeric Aβ1-42. 5.6.3 Example 3 Binding of oligomeric Aβ1-42 to CD22-expressing cells.

[0273] HEK293 cells were cultured at 31,250 cells / cm on day 1. 2 HEK293 cells were seeded on 6-well plates at a density of 1000 x 1000 (Invitrogen) on day 2 to overexpress full-length human CD22 for 2 days. On day 4, the transfected cells were incubated with 1 μg / mL FITC-Aβ in growth medium on ice for 1 h. The cells were trypsinized and analyzed by flow cytometer. As shown in Figure 2, overexpression of human CD22 in HEK293 cells increased the binding of FITC-Aβ onto the surface of HEK293 cells, demonstrating the binding between Aβ and CD22 on the cell surface. 5.6.4 Example 4 CD22 binding to oligomeric Aβ1-42 on HMC-3 cells

[0274] Proximity ligation assay (PLA) was used to assess binding of CD22 to Aβ1-42 on HMC-3 cells. HMC-3 cells were plated on 1% gelatin-coated coverslips at 26,000 cells / cm. 2 The cells were seeded at a density of 1000 x 1000. The cells were treated with oligomeric Aβ on ice for 1 h. The cells were washed once with PBS and then fixed with 4% paraformaldehyde (PFA) for 5 min. The cells were incubated with anti-CD22 and anti-Aβ antibodies at 4°C overnight. The PLA assay was performed according to the manufacturer's protocol (Duolink® Proximity Ligation Assay, Sigma-Aldrich). Fluorescent images were acquired using a Zeiss 710 upright microscope. As shown in Figure 3, HMC-3 cells were surrounded by a dotted line. The positive signal indicated by the arrow demonstrated the physical interaction between CD22 and Aβ. 5.6.5 Example 5 Anti-CD22 antibody induced internalization of CD22

[0275] HMC3 cells were plated on 1% gelatin-coated coverslips at 26,000 cells / cm 2 The cells were seeded at a density of 10 μg / mL. The cells were treated with 10 μg / mL SM03 and incubated on ice for 1 hour. The cells were then incubated at 37° C. to induce internalization. At the indicated time points, the medium was removed and the cells were fixed with 4% PFA. Surface CD22 expression was detected using mouse anti-human CD22 antibody (Abcam) and Alexaflour488-conjugated anti-mouse IgG antibody. As shown in FIG. 4, HMC-3 cells were treated with SM03 for 3, 5, 10, 30, and 60 minutes. SM03 reduced surface CD22 expression by 20% only 3 minutes after treatment. 5.6.6 Example 6 Internalization of anti-CD22 antibody in human microglial cells.

[0276] HMC-3 cells were plated on 1% gelatin-coated coverslips at 26,000 cells / cm2 HMC3 cells were seeded at a density of 100x and treated with FITC-SM03 for 30 minutes. HMC3 cells were fixed with 4% paraformaldehyde in PBS, and confocal images were acquired using a Zeiss 800 microscope. As shown in Figure 5, FITC-SM03 was internalized in HMC3 cells within 30 minutes after treatment.

[0277] HMC-3 cells were cultured at 30,000 cells / cm 2 Cells were seeded on 6-well plates at a density of 10 μg / mL. Cells were incubated with the indicated anti-CD22 antibodies at 10 μg / mL on ice for 2 hours. Cells were then incubated at 37° C. to induce internalization. At the indicated time points, the medium was removed and cells were fixed with 4% PFA. Surface anti-CD22 antibodies, i.e., SM03, SM06, Emab or M971, were each detected with an AlexaFlour 488-conjugated anti-human IgG antibody (Invitrogen). As shown in FIG. 6, different anti-CD22 antibodies showed different rates of internalization by HMC-3 cells. SM03 and SM06 induced the highest rate of internalization by HMC-3 cells. 5.6.7 Example 7 Anti-CD22 antibody enhances internalization of oligomeric Aβ

[0278] HMC-3 cells were cultured at 60,000 cells / cm 2HMC-3 cells were seeded on 6-well plates at a density of 1 μg / mL. HMC-3 cells were pre-treated with 1 μg / mL FITC-Aβ in growth medium for 24 hours. Cells were then treated with 10 μg / mL SM03, SM06, Emab and M971 or IgG isotype control for the indicated times, e.g., 4 hours, 8 hours, 16 hours and 24 hours. Cells were trypsinized and fixed with 4% PFA at room temperature for 5 minutes. Cells were stained with mouse anti-human CD22 antibody (Abcam) and FITC-Aβ internalization was analyzed using a flow cytometer. As shown in FIG. 7, SM03, SM06 and Emab (but not M971) showed enhanced phagocytosis of FITC-Aβ. Both SM03 and SM06 showed the highest phagocytosis ability of all antibodies tested. 5.6.8 Example 8 Anti-CD22 antibody suppressed the NFκB signaling pathway in microglial cells

[0279] HMC-3 cells were cultured at 50,000 cells / cm 2 Cells were seeded on 24-well plates at a density of 1000 x 1000. Cells were transfected with an NFκB luciferase reporter plasmid (Invitrogen, Lipofectamine 3000). The transfected cells were then stimulated with LPS and co-treated with different concentrations of SM03 Fab fragments. Luciferase readings were recorded 24 hours after treatment. As shown in Figure 8, SM03 Fab reduced NFκB signaling in a dose-dependent manner, demonstrating that microglial activation was reduced by SM03 treatment. 5.6.9 Example 9 Anti-CD22 antibody suppressed IL-6 secretion in microglial cells

[0280] HMC-3 cells were cultured at 125,000 cells / cm 2HMC-3 cells were stimulated with LPS and co-treated with different concentrations of SM03 Fab fragments. After 24 hours of treatment, the medium was collected and tested using an IL-6 ELISA kit (R&D systems) according to the manufacturer's protocol. As shown in Figure 9, SM03 Fab reduced IL-6 secretion, further confirming that microglial activation was reduced with SM03 treatment. 5.6.10 Example 10 Anti-CD22 antibody promoted trans-binding of 2,6-sialic acid on HMC-3 cells

[0281] HMC3 cells were plated on 1% gelatin-coated coverslips at 26,000 cells / cm 2 Cells were seeded at a density of 100-1500 nm. Cells were treated with anti-CD22 antibody for the indicated times. The medium was removed and cells were fixed with 4% PFA at room temperature for 5 min. Fixed cells were incubated with FITC-conjugated 2,6-sialic acid probe (GlycoTech) at 4° C. overnight. Fluorescent images were acquired using a Leica DMI6000B microscope. As shown in FIG. 10, SM03 and SM06 promoted trans-binding of 2,6-sialic acid probe in HMC-3 cells. 5.6.11 Example 11 Anti-CD22 antibodies reduce synaptic phagocytosis and neuronal death

[0282] Anti-CD22 antibodies, including SM03 and SM06, are subjected to functional assays to test their activity in reducing synaptic phagocytosis and / or neuronal death. To evaluate synaptic phagocytosis, HMC-3 cells are co-cultured with differentiated SH-SY5Y neuronal cells. Synapses developed on differentiated SH-SY5Y are indicated by the expression of PSD-95 protein. PSD-95 expression in HMC-3 microglial cells can be measured using Western blot or ELISA, which shows the rate of synaptic phagocytosis by microglial cells. Treatment with SM03 and / or SM06 reduces PSD-95 expression in HMC-3 cells, indicating that these anti-CD22 antibodies can inhibit synaptic phagocytosis by microglial cells.

[0283] To measure neuronal death, differentiated SH-SY5Y cells are treated with the culture medium of HMC-3 cells after LPS stimulation in the presence or absence of the indicated anti-CD22 antibodies. The death of SH-SY5Y is measured by MTT assay. Treatment with SM03 and / or SM06 reduces the death of SH-SY5Y. 6. REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTINGS

[0284] This application incorporates by reference the Sequence Listing entitled "022A003WO01.XML", which was created on July 1, 2022 and has a size of 56,981 bytes.

Claims

**Claim 1** A composition for use in promoting the removal of beta-amyloid (Aβ) plaques or reducing neuroinflammation in a subject in need of promoting the removal of beta-amyloid (Aβ) plaques or reducing neuroinflammation, the composition comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (a) promotes the cis-trans conversion of CD22 and / or (b) induces the internalization of CD22. **Claim 2** The composition according to claim 1, wherein the subject has clinical or preclinical Alzheimer's disease, prodromal Alzheimer's disease, Down syndrome, clinical or preclinical cerebral amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia, cognitive impairment, memory loss, or vascular disorders caused by pathogenic Aβ peptides in the blood vessels. **Claim 3** A composition for use in treating the same in a subject in need of treating an Aβ-related or neuroinflammation-related disease or disorder, the composition comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (a) promotes the cis-trans conversion of CD22 and / or (b) induces the internalization of CD22. **Claim 4** The composition according to claim 3, 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 cerebral amyloid angiopathy (CAA), Parkinson's disease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia, cognitive impairment, memory loss, or vascular disorders caused by pathogenic Aβ peptides in the blood vessels. **Claim 5** The composition according to claim 3, wherein the Aβ-related or neuroinflammation-related disease or disorder is Alzheimer's disease. **Claim 6** The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. **Claim 7** The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is an antibody selected from the group consisting of IgG1 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody, or an antibody or antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single domain antibody (sdAb), and heavy chain antibody (HCAb).

8. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

9. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment specifically binds to CLLNFSCYGYP IQ (SEQ ID NO: 11) and VFTRSELKFS PQWSHHGKIVT C (SEQ ID NO: 12) of human CD22.

10. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having the 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 the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

11. The composition according to claim 10, wherein the antibody or antigen-binding fragment is a chimeric antibody or antigen-binding fragment, and 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.

12. The composition according to claim 10, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment, and 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.

13. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is administered in the range of 1 to 50 mg per kg of the body weight of the subject.

14. The composition according to claim 13, wherein the antibody or antigen-binding fragment is administered at about 1, about 2, about 3, about 5, about 10, about 15, or about 30 mg per kg of the body weight of the subject.

15. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is administered at 300 to 1,200 mg per dose.

16. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is administered once every two weeks or once a month.

17. The composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is administered in multiple doses over a period of at least 3 months, at least 6 months or at least 1 year.

18. The composition according to any one of claims 1 to 5 for use in combination with a second therapeutic agent.

19. The composition according to claim 18, wherein the second therapeutic agent is an anti-beta-amyloid antibody, an anti-CD33 antibody, a tau aggregation inhibitor, a tau protein modulator, a cholinesterase inhibitor, an acetylcholinesterase inhibitor, an N-methyl D-aspartic acid (NMDA) antagonist, a beta-secretase inhibitor or an insulin sensitivity improver.

20. The composition according to any one of claims 1 to 5, wherein the subject is a human subject.

21. A composition for use in inducing the internalization of Aβ by microglial cells, comprising an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (a) promotes the cis-trans conversion of CD22 and / or (b) induces the internalization of CD22.

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