Anti-siglec compositions and uses thereof for treating neurodegenerative diseases

Abstract

Provided herein are anti-Siglec-10 and Siglec-8 antibody compositions and combinations thereof, and their use in the treatment of neurodegenerative diseases.

Description

Attorney Docket No. 111005.0901.01PC00ANTI-SIGLEC COMPOSITIONS AND USES THEREOF FOR TREATING NEURODEGENERATIVE DISEASESFIELD OF THE INVENTION

[0001] The present invention relates to anti-Siglec-10 and anti-Siglec-8 antibody compositions and combinations thereof, and their use in the treatment of neurodegenerative diseases.BACKGROUND OF THE INVENTION

[0002] Members of the CD33 family of Siglec proteins consist of one or more IgV-like domains and one or more IgC-like domains, a transmembrane domain and intracellular domains with two immunoreceptor tyrosine-based inhibitory motives (“ITIMs”). The IgV-like domain confers them with reactivity to sialylated glycans, while the HIM suggest their function as inhibitory receptors for certain physiological functions. Due to lack of endogenous ligand for Siglecs, the biological function of the CD33 family was unknown until 2009, when Chen et al. reported CD24 as the endogenous ligand for Siglec 10 to mediate negative regulatory for innate immune response to tissue injuries, thus establishing CD24-Siglec 10 interaction as an immune checkpoint for innate immunity. In 2019, Barkal et al extended this pathway to regulation of phagocytosis of cancer cells.

[0003] Meanwhile, genetic studies suggest that single-nucleotide polymorphism (“SNP”) is associated with a higher risk of Alzheimer’s disease (“AD”) development. Further study suggested that an antibody against mouse CD22, a member of CD33 -family of Siglecs, may potentially regulate microglia function and thus may be potentially useful for treatment of AD. However, since CD22 is not expressed in human microglia, the significance of these findings for human AD is less clear.

[0004] The present inventors have now found that transgenic expression of human Siglec- 10 and Siglec-8 exacerbates the pathological changes associated with development of AD, and that antagonist antibodies against Siglec-10 promote the proper function of the microglia in its phagocytosis of fibrins and ameliorates pathological changes in mouse AD models.

[0005] Accordingly, there is a need in the art for new immunotherapies that target the Siglec-10 proteins for treating neurodegenerative diseases.-1-105756061.1Attorney Docket No. 111005.0901.01PC00SUMMARY OF THE INVENTION

[0006] Provided herein is a method of treating a neurodegenerative disease in a subject in need thereof, which may comprise administering an anti-Siglec-10 antibody to the subject. As described herein the anti-Siglec-10 antibody may be an antigen binding fragment thereof. Further provided are the anti-Siglec-10 antibody for treating the neurodegenerative disease and use of the anti-Siglec-10 antibody in the manufacture of a medicament for treating the neurodegenerative disease. The neurodegenerative disease may be selected from Alzheimer’s diseases, dementia, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease. In one aspect, the neurodegenerative disease is Alzheimer's disease.

[0007] The anti-Siglec-10 antibody may ameliorate the accumulation of AP amyloid in the brain. The anti-Siglec-10 antibody may ameliorate the accumulation of phosphorylated Tau protein in the brain. The anti-Siglec-10 antibody may further enhance phagocytosis of amyloid or phosphorylated Tau fibrins by microglia. The anti-Siglec-10 antibody may penetrate CNS and occupy Siglec-10 receptor on the microglia.

[0008] The anti-Siglec-10 antibody may comprise (a) a heavy chain variable region comprising one or more of a complementarity determining region (CDR) 1 comprising the sequence set forth in SEQ ID NO: 3, a CDR2 comprising the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising the sequence set forth in SEQ ID NO: 5; and, (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the sequence set forth in SEQ ID NO: 8. The anti-Siglec-10 antibody may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 2. The anti-Siglec-10 antibody may be 3 IF 11.

[0009] The anti-Siglec-10 antibody may comprise a heavy chain variable region comprising the sequence set forth in one of SEQ ID NOS: 9-13, and the light chain variable region may comprise the sequence set forth in one of SEQ ID NOS: 14-18. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 9, 10, or 11, and the light chain variable region may comprise the sequence set forth in SEQ ID NO: 15. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 9. The anti-Siglec-10 antibody may comprise the heavy chain comprising the heavy chain variable region comprising the sequence-2-105756061.1Attorney Docket No. 111005.0901.01PC00 set forth in SEQ ID NO: 9 and a constant region comprising a human IgG4. The human IgG4 may comprise the sequence set forth in SEQ ID NO: 21.

[0010] The anti-Siglec-10 antibody may comprise the heavy chain comprising the sequence set forth in SEQ ID NO: 9 and the light chain variable region may comprise the sequence set forth in SEQ ID NO: 15. The anti-Siglec-10 antibody may comprise a heavy chain comprising the sequence set forth in SEQ ID NO: 22 and a light chain comprising the sequence set forth in SEQ ID NO: 23. The anti-Siglec-10 antibody may be ONC-841.

[0011] The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 10. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 11. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 10 or 12, and the light chain variable region may comprise the sequence set forth in SEQ ID NO: 17. The heavy chain variable region may comprise the sequence set forth in SEQ NO: 10. The heavy chain variable region may comprise the sequence set forth in SEQ NO: 12. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 10 or 12, and the light chain variable region may comprise the sequence set forth in SEQ ID NO: 16. The heavy chain variable region may comprises the sequence set forth in SEQ ID NO: 10. The heavy chain variable region may comprise the sequence set forth in SEQ ID NO: 12.

[0012] Also provided herein is an anti-Siglec-8 antibody. As described herein the anti-Siglec-8 antibody may be an antigen binding fragment thereof. The anti-Siglec-8 antibody may comprise: (a) a heavy chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 26 or 27; a CDR2 comprising the sequence set forth in SEQ ID NO: 28 or 29; and a CDR3 comprising the sequence set forth in SEQ ID NO: 30; and (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 31 or 32; a CDR2 comprising the sequence set forth in SEQ ID NO: 33; and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or 35.

[0013] The anti-Siglec-8 antibody may comprise the heavy chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 26, and the CDR2 comprising the sequence set forth in SEQ ID NO: 28; and the light chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 31, and the CDR3 comprising the sequence set forth in SEQ ID NO: 34. The anti-Siglec-8 antibody may comprise a heavy chain comprising the heavy chain variable region and a human IgG4 constant region comprising the sequence set forth-3-105756061.1Attorney Docket No. 111005.0901.01PC00 in SEQ ID NO: 36. Also provided herein is a pharmaceutical composition comprising the anti- Siglec-8 antibody and one or more pharmaceutically acceptable carriers or excipients.

[0014] The anti-Siglec-10 antibody may comprise a bi-specific antibody. The anti-Siglec-10 antibody further may comprise an anti-Siglec-8 antibody domain. The anti-Siglec-8 antibody domain may comprise the anti-Siglec-8 antibody. The anti-Siglec-8 antibody domain may comprise Lirentelimab, 7C9, or an antibody binding fragment thereof.

[0015] Further provided herein is a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering the anti-Siglec-8 antibody to the subject. Also provided are the anti-Siglec-8 antibody for treating the neurodegenerative disease and use of the anti-Siglec-8 antibody in the manufacture of a medicament for treating the neurodegenerative disease. The neurodegenerative disease may comprise Alzheimer’s disease, dementia, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), or Creutzfeldt- Jakob disease. The neurodegenerative disease may comprise Alzheimer’s disease. The anti-Siglec-8 antibody may ameliorate the accumulation of A0 amyloid in the brain. The anti-Siglec-8 antibody may also ameliorate the accumulation of phosphorylated Tau protein in the brain. The anti-Siglec-8 antibody may enhance phagocytosis of amyloid or phosphorylated Tau fibrins by microglia. The anti-Siglec 8 antibody may penetrate CNS and occupy Siglec-8 on the microglia.

[0016] Provided herein is also a method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-10 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.

[0017] Provided herein is also a method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-8 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.

[0018] Provided herein is also a method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-10 and Siglec-8 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of-4-105756061.1Attorney Docket No. 111005.0901.01PC00 phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.

[0019] The mouse may comprise a genomic clone of human Siglec genes, comprising at least Siglec-8 and Siglec- 10 coding and regulatory sequences.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office by request and payment of the necessary fee.

[0021] FIG. 1 describes the generation of a monoclonal anti-Siglec-10 antibody. The figure shows the antagonist activity of anti-Siglec-10 antibodies in a reporter assay for antibodydependent cell-mediated cytotoxicity (ADCC). Grading doses of 4 different anti-Siglec-10 antibodies were compared for their ability to rescue ADCC suppressed by Siglec- 10 ectopically expressed in Jurkat ADCC effector cells. Target: CHO-CTLA4-OFP clone 9 (2xl04). Effector: Jurkat-PWPI-SigleclO (2xl04), ADCC-triggering antibody, anti-CTLA4 mAb HL32: (0.1 pg / ml).

[0022] FIG. 2A-B show Siglec transgenic mice recapitulating microglia-specific expression of Siglec-8 and Siglec-10. FIG. 2A. In silico analysis of single-cell RNA sequencing data showing microglia specific expression of Siglec-8 and Siglec-10 genes. FIG. 2B. Flow cytometry profile of Siglec-10 and Siglec-8 expression specifically on microglia among a single cell suspension of transgenic mouse brain.

[0023] FIG. 3A-D show the increased accumulation of A0 amyloid plaques (FIG. 3A and B) and phosphorylated Tau protein (FIG. 3C and D) as revealed by immunohistochemistry using antibody specific for AA 707-713 of A0 amyloid protein (FIG. 3 A and B) or anti-phosphorylated Tau (“AT8”) protein (FIG. 3C and D).

[0024] FIG. 4A-E show that Anti-Siglec-10 antibody 31F11 reduces accumulation of pathogenic A0 plaque and phosphorylated Tau proteins. FIG. 4A. Study schedule and quantitative analysis of amyloid A0 plaque number and intensity. FIG. 4B. Representative high-power images of anti- A0 amyloid staining of brain section. FIG. 4C. Quantitative analysis of pTau intensity in cortex and hippocampus. FIG. 4D and E. Representative images of pTau staining in cortex and hippocampus.-5-105756061.1Attorney Docket No. 111005.0901.01PC00

[0025] FIG. 5A-C show that Anti -Si glee- 10 monoclonal antibody ameliorates Ab amyloid accumulation in the Siglec-APP transgenic mice (Taconic ARTE10 mouse model 16347, B6.CBA-Tg(Thyl-PSENl*M146V,-APP*Swe)10Arte). FIG. 5A. Treatment regimen (left) and low power images of PBS (middle) or anti-Siglec-10 mAb-treated mouse brain section (right). FIG. 5B. High power images of representative cortex staining. FIG. 5C. Confirmation of target blocking by anti-Siglec-10 mAb.

[0026] FIG. 6A-C show that Anti-Siglec-10 monoclonal antibody reduced plasma levels of mutant Tau and phosphorylated Tau proteins. SIGLEC x Pmp-MAPT*P 30 IL Fl transgenic mice were treated with anti-SIGLEC 10 mAb 31F11 for 6.5 weeks (FIG. 6A) and the levels of mutant human Tau (FIG. 6B) and phosphorylated Tau (pTau181) (FIG. 6C) were measured.

[0027] FIG. 7A-E show that ONC-841 effectively blocked Siglec-10 in the Siglec TG brain. FIG. 7A. Diagram of experimental design. FIG. 7B. Representative FACS profile. FIG. 7C. Quantitative analysis of Siglec-10 binding intensity, with background staining subtracted.FIG. 7D. Immunohistochemistry staining demonstrating blocking of cell surface Siglec-10.FIG. 7E. Immunohistochemistry staining demonstrating normal microglia number after chronic anti-Siglec 10 treatment.

[0028] FIG. 8A-C show that ONC-841 promotes phagocytosis of Ap fibrin by Siglec TG mouse microglia. A.) Diagram of study design. B.) Representative FACS profile. C.) Dose dependence and EC50.

[0029] FIG. 9A-C shows that ONC-841 promotes phagocytosis of phosphorylated Tau fibrin by Siglec TG mouse microglia. FIG. 9A. Diagram of study design. FIG. 9B. Representative FACS profile. FIG. 9C. Dose dependence and EC50.

[0030] FIG. 10A-C show that ONC-841 promotes phagocytosis of Tau-PFF by human microglia. FIG. 10A. Diagram of study design. FIG. 10B. Representative FACS profile. FIG. 10C. Dose dependence and EC50.

[0031] FIG. 11A-C show that ONC-941m promotes phagocytosis of Ap fibrin by Siglec TG mouse microglia. FIG. 11 A. Diagram of study design. FIG. 1 IB. Representative FACS profile. FIG. 11C. Dose dependence and EC50.

[0032] FIG. 12A-C show that ONC-941m promotes phagocytosis of Tau-PFF by human microglia. FIG. 12A. Diagram of study design. FIG. 12B. Representative FACS profile. FIG. 12C. Dose dependence and EC50.-6-105756061.1Attorney Docket No. 111005.0901.01PC00

[0033] FIG. 13 shows that 31F11 significantly reduces amyloid plaques in aged mice.

[0034] FIG. 14 shows quantification of amyloid plaques in SiglO / ARTE mice.

[0035] FIG. 15 shows representative high-power images of Glial Fibrillary Acidic Protein (GFAP) from PBS (left) or 31F11 treated 13 month old siglecg KO / SigleclO ARTE mice.

[0036] FIG. 16 shows quantification of astrocyte average intensity.

[0037] FIG. 17 shows brain sections stained with anti-IBAl antibody (EPR16588) from PBS (left) or 3 IF 11 (right) treated 13 month old siglecg KO / SigleclO / ARTE mice.

[0038] FIG. 18 shows the representative NeuN staining from the hippocampus CAI area from PBS (left) or 31F11 (right) treated mice. 31F11 significantly rescued neuronal loss and degeneration.

[0039] FIG. 19A shows an elevated plus maze (EPM).

[0040] FIG. 19B shows that 31F11 treated mice spent considerably less time in the open arms of the EPM compared to the PBS treated mice.

[0041] FIG. 19C shows mice treated with 31F11 showed a significant improvement in time in the open arms of the EPM as compared to PBS-treated mice.

[0042] FIG. 20A shows the novel object recognition test (NORT).

[0043] FIG. 20B shows that 31F11 treated mice display better preference to the novel object in the NORT, indicating improved memory related functions following treatment.

[0044] FIG. 21 A shows representative images of brain sections stained for A|3 from mice treated with 31F11.

[0045] FIG. 2 IB shows a significant decrease in average plaque area and a tendency of reduced plaque numbers following anti-SigleclO treatment.

[0046] FIG. 22A shows representative high-power images of IBA1 from PBS (left) or 3 IF 11 (right) treated siglecg KO / SigleclOIARTE mice.

[0047] FIG. 22B shows the quantification of the number of processes of each microglia.

[0048] FIG. 23A shows representative high-power images of GFAP from PBS (left) or ONC- 841 (right) treated siglecg KO / SigleclO / ARTE mice.

[0049] FIG. 23B shows quantification of astrocyte average intensity.-7-105756061.1Attorney Docket No. 111005.0901.01PC00DETAILED DESCRIPTION

[0050] Provided herein are anti-Siglec-10 antibody and anti-Siglec-8 antibody compositions of matter and antigen binding fragments thereof. Each antibody may be a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody. Each antibody may be monospecific, bispecific, trispecific or multispecific. The anti-Siglec-10 or anti-Siglec-8 antibody may comprise an antigen-binding fragment of an antibody that immunospecifically binds to one or more of Siglec-10 and Siglec-8, which in particular may be expressed on the surface of a live cell at an endogenous or transfected concentration. Also provided are antibody molecules of which the antigen-binding fragment binds to Siglec-10 or Siglec-8. Each antibody may be detectably labeled or comprise a conjugated toxin, drug, receptor, enzyme, or receptor ligand.

[0051] Responsiveness of different tumor types and patients varies, which means that different inhibitory pathways may be relevant to different neurodegenerative diseases or different patient populations. Therefore, the identification of additional do-not-eat-me signals can lead to the development of new therapies that are more effective for the treatment of neurodegenerative disease, either alone or possibly in combination. The antibodies described herein may be used to treat cancers by administering them as described herein, either alone or in combination with other therapies.

[0052] The inventors have discovered that anti-Siglec-10 and anti-Siglec-8 antibodies exhibit surprisingly potent binding to Siglec-10 and Siglec-8, respectively, particularly on the cell surface. The antibodies also exhibit surprisingly strong activity in the treatment of neurodegenerative disease.1. Definitions

[0053] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. The word “about” in association with a numeric value denotes a reasonable approximation of that value. In certain cases “about” may be construed as being within as much as 10% of the specific value with which it is associated. For example, the phrase “about 100” would encompass any value between 90 and 110.-8-105756061.1Attorney Docket No. 111005.0901.01PC00

[0054] For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0055] “ Treatment” or “treating,” when referring to protection of an animal from a disease, means preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a composition of the disclosure to an animal prior to onset of the disease. Suppressing the disease involves administering a composition of the disclosure to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a composition of the disclosure to an animal after clinical appearance of the disease. Treating the disease may refer to reducing one or more symptoms of the disease.

[0056] As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelid antibodies, single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies disclosed herein). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG2, IgG.3, IgG , IgAi and IgA2) or subclass.

[0057] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody's CDR and optionally the framework residues-9-105756061.1Attorney Docket No. 111005.0901.01PC00 that comprise the antibody's “variable region” antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins comprising the antibody's “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0058] Human, chimeric or humanized antibodies are particularly preferred for in vivo use in humans, however, murine antibodies or antibodies of other species may be advantageously employed for many uses (for example, in vitro or in situ detection assays, acute in vivo use, etc.).

[0059] A “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, the contents of each of which are incorporated herein in their entirety), veneering or resurfacing (EP 592,106; EP 519,596, the contents of each of which are incorporated herein by reference), and chain shuffling (U.S. Pat. No. 5,565,332, the contents of which are incorporated herein by reference).

[0060] Also contemplated herein are “humanized antibodies.” As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. The non-human-10-105756061.1Attorney Docket No. 111005.0901.01PC00 immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. The donor antibody may be referred to as having been “humanized,” by the process of “humanization,” because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an FcyRIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).2. Anti-Siglec-10 antibody compositions

[0061] Provided herein is an anti-Siglec-10 antibody or antigen binding fragment thereof. It is understood that one more features of the antibody described herein may also be included in an antigen binding fragment. The anti-Siglec-10 antibody may be a monoclonal antibody, single chain antibody, a bi-specific antibody, tri-specific antibody, multi-specific antibody, or chimeric-11-105756061.1Attorney Docket No. 111005.0901.01PC00 antibody. The anti-Siglec 10 antibody may be as described in U.S. Patent Application Publication No. 2024 / 0218067, the contents of which are incorporated herein in their entirety.

[0062] The anti-Siglec- 10 antibody may comprise one or more sequences of antibody 31F11, which comprises heavy and light chain variable regions comprising the sequences set forth in SEQ ID NOs: 1 and 2, respectively. The antibody may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 1, and may comprise a light chain variable region comprising the sequence set forth in SEQ ID NO: 2. The heavy chain variable region of the anti-Siglec-10 antibody may comprise one or more of: a CDR1 comprising the sequence set forth in SEQ ID NO: 3, a CDR2 comprising the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising the sequence set forth in SEQ ID NO: 5. The light chain variable region of the anti-Siglec-10 antibody may comprise one more of: a CDR1 comprising the sequence set forth in SEQ ID NO: 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the sequence set forth in SEQ ID NO: 8.

[0063] In one example, the heavy chain variable region comprises CDR1-3 having SEQ ID NOs: 3-5, respectively. In another example, the light chain variable region comprises CDR1-3 having SEQ ID NOs: 6-8, respectively. In a further example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising SEQ ID NOs: 3-5 and the light chain variable region comprising SEQ ID NOs: 6-8.

[0064] One or more of the heavy and light chains of the anti-Siglec-10 antibody may also be humanized relative to 31F11. The anti-Siglec-10 antibody may comprise one or more heavy chain variable regions, each comprising the sequence set forth in one of SEQ ID NOS: 9-13 (named hu-VHvl, VHv2, VHv3, VHv4, and VHv5, respectively). The anti-Siglec-10 antibody may comprise one or more light chain variable regions, each comprising the sequence set forth in one of SEQ ID NOS: 14-18 (named hu-VLvl, VLv2, VLv3, VLv4, and VLv5, respectively).

[0065] In one example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 9 or the heavy chain comprising the sequence set forth in SEQ ID NO: 22, and the light chain variable region comprising the sequence set forth in SEQ ID NO: 15 or the light chain comprising the sequence set forth in SEQ ID NO: 23. The anti-Siglec-10 antibody may comprise hu-VHvlVLv2.

[0066] In another example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 10 or the heavy chain comprising the-12-105756061.1Attorney Docket No. 111005.0901.01PC00 sequence set forth in SEQ ID NO: 24, and the light chain variable region comprising the sequence set forth in SEQ ID NO: 15 or the light chain comprising the sequence set forth in SEQ ID NO: 23. The anti-Siglec-10 antibody may comprise hu-VHv2VLv2.

[0067] In a further example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 10 or the heavy chain comprising the sequence set forth in SEQ ID NO: 24, and the light chain variable region comprising the sequence set forth in SEQ ID NO: 16 or the light chain comprising the sequence set forth in SEQ ID NO: 25. The anti-Siglec-10 antibody may comprise hu-VHv2VLv3.

[0068] The anti-Siglec-10 antibody may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 9, 10, or 11; and the light chain variable region comprising the sequence set forth in SEQ ID NO: 15, 23, 16 or 25.

[0069] In another example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 10 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 17. The anti-Siglec-10 antibody may comprise hu-VHv2VLv4. In a further example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 12 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 17. The anti-Siglec-10 antibody may comprise hu-VHv4VLv4. The anti-Siglec-10 antibody may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 10 or 12; and the light chain variable region comprising the sequence set forth in SEQ ID NO: 17.

[0070] In another example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 12 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 16 or 25. The anti-Siglec-10 antibody may comprise hu-VHv4VLv3.

[0071] In another example, the anti-Siglec-10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 10; and the light chain variable region comprising the sequence set forth in SEQ ID NO: 15 or 17. In a further example, the anti-Siglec- 10 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 12; and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16, 25, or 17.-13-105756061.1Attorney Docket No. 111005.0901.01PC00

[0072] The anti-Siglec-10 antibody may comprise a human IgK polypeptide. In one example, the IgK has the sequence set forth in SEQ ID NO: 19. The anti-Siglec-10 antibody may comprise a human IgG constant region, which may be IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2. In one example, the IgG constant region is IgG4, which may have the sequence set forth in SEQ ID NO: 20. The IgG4 constant region may comprise a S108P substitution relative to the sequence of SEQ ID NO: 20, and may comprise the sequence set forth in SEQ ID NO: 21. In another example, the anti-Siglec-10 antibody comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 9 and the mutant IgG4 constant region comprising the sequence set forth in SEQ ID NO: 21.

[0073] The anti-Siglec-10 antibody may comprise the heavy chain comprising the sequence set forth in SEQ ID NO: 9 or 22, and the light chain comprising the sequence set forth in SEQ ID NO: 15 or 23 (VHvlVLv2). In one example, the anti-Siglec-10 antibody comprises the heavy chain comprising the sequence set forth in SEQ ID NO: 22 and the light chain comprising the sequence set forth in SEQ ID NO: 23, and may be referred to as ONC-841.

[0074] The anti-Siglec-10 antibody may comprise the heavy chain comprising the sequence set forth in SEQ ID NO: 10 or 24, and the light chain comprising the sequence set forth in SEQ ID NO: 15 or 23 (VHv2VLv2). In one example, the antibody comprises the heavy chain comprising the sequence set forth in SEQ ID NO: 24 and the light chain comprising the sequence set forth in SEQ ID NO: 23. The anti-Siglec-10 antibody may comprise the heavy chain comprising the sequence set forth in SEQ ID NO: 10 or 24, and the light chain comprising the sequence set forth in SEQ ID NO: 16 or 25 (VHv2VLv3). In one example, the antibody comprises the heavy chain comprising the sequence set forth in SEQ ID NO: 24 and the light chain comprising the sequence set forth in SEQ ID NO: 25.3. Anti-Siglec-8 antibody compositions

[0075] Provided herein is an anti-Siglec-8 antibody or antigen binding fragment thereof. It is understood that one more features of the antibody described herein may also be included in an antigen binding fragment. The anti-Siglec-8 antibody may be a monoclonal antibody, single chain antibody, a bi-specific antibody, tri-specific antibody, multi-specific antibody, or chimeric antibody. The anti-Siglec-8 antibody may comprise a heavy chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 26, a CDR2 comprising the sequence set forth in SEQ ID NO: 28, and a CDR3 comprising the sequence set forth in SEQ ID NO: 30.-14-105756061.1Attorney Docket No. 111005.0901.01PC00The anti-Siglec-8 antibody may comprise a light chain variable region comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 31, a CDR2 comprising the sequence set forth in SEQ ID NO: 33, and a CDR3 comprising the sequence set forth in SEQ ID NO: 34. The anti- Siglec-8 antibody may be referred to as ONC-941m.

[0076] The anti-Siglec-8 antibody may be a humanized variant of ONC-941m comprising at least one humanized CDR relative to a corresponding CDR in ONC-941m. The anti-Siglec-8 antibody may comprise a heavy chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 26 or 27; a CDR2 comprising the sequence set forth in SEQ ID NO: 28 or 29; and a CDR3 comprising the sequence set forth in SEQ ID NO: 30. The humanized variant of ONC-941m may comprise a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 31 or 32; a CDR2 comprising the sequence set forth in SEQ ID NO: 33; and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or 35.

[0077] The heavy chain of the anti-Siglec-8 antibody may comprise an IgG constant region as disclosed herein above. The IgG constant region may comprise human IgG4 constant region or a mutant variant thereof. The human IgG4 constant region may comprise the following sequence. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GL YSL S S VVTVP S S SLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPCP APEFLGGP S V FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVLHEALHSHYTQKSLSLSLGK (SEQ ID NO: 36)4. Bi-specific antibody compositions

[0078] The anti-Siglec-10 antibody or anti-Siglec-8 antibody may be a bi-specific antibody. The bi-specific antibody may comprise a second antibody or antigen binding fragment thereof. The second antibody or antigen binding fragment thereof may target an anti-tumor pathway or mechanism complementary to one targeted by the anti-Siglec-10 antibody or anti-Siglec-8 antibody. In one example, an anti-Siglec-10 antibody composition described herein is combined with an anti-Siglec-8 antibody disclosed herein, which may further enhance the treatment of neurodegenerative diseases. Thus, in one example the bi-specific antibody comprises the anti--15-105756061.1Attorney Docket No. 111005.0901.01PC00Siglec-10 antibody and the anti-Siglec-8 antibody, or one or more antigen binding fragments thereof disclosed herein.

[0079] The bi-specific anti-Siglec-10 antibody may comprise an anti-Siglec-8 antibody domain. The anti-Siglec-8 antibody domain may comprise Lirentelimab (AK002) or an antigen binding portion thereof. In one example, the anti-Siglec-8 antibody domain comprises clone 7C9 (which may be sold by BIOLEGEND), which is a mouse antibody consisting of an IgGl heavy chain and a kappa light chain. In yet a further example, the anti-Siglec-8 antibody domain comprises ONC-941m, a humanized variant thereof, or an antigen binding fragment of the foregoing.

[0080] There are many different bi-specific antibody technologies known in the art. Most of these require that the two-component antibodies are in a single chain format so that the two parts can be expressed in a single construct. One exemplary method is to express the antibodies as a single-chain variable fragment (scFv). Non-limiting examples of bi-specific antibody technologies include BiTE (for Bi-specific T-cell Engager), DART (for Dual-Affinity ReTargeting), Fabs-in-tandem immunoglobulin (FIT-Ig), and knobs-into-holes.

[0081] Methods of Treatment

[0082] Provided herein are uses of the antibody compositions described herein, and pharmaceutical compositions thereof, for treating a neurodegenerative disease. As used herein, the term “neurodegenerative disease” refers to disorders that are primarily characterized by neuron loss over time. Neurodegenerative diseases include, but are not limited to, Alzheimer’s disease, dementia, Parkinson’s disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, motor neuron disease, Huntington’s disease, etc. The neurodegenerative disease may be Alzheimer’s disease, dementia, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), or Creutzfeldt-Jakob disease.

[0083] Provided herein is a method of treating the neurodegenerative disease in a subject in need thereof, which may comprise administering the antibody composition to the subject. The subject may be a mammal, such as a dog, cat, pig, horse, cow, monkey, ape, or human. In one example, the subject is a human patient. Also provided is the antibody composition for use in treating the neurodegenerative disease. Further provided is use of the antibody composition in the manufacture of a medicament for treating the neurodegenerative disease. In one example, the antibody composition is used or intended for use as monotherapy, which may facilitate the amelioration of the neurodegenerative disease.-16-105756061.1Attorney Docket No. 111005.0901.01PC00

[0084] In another embodiment, the antibody composition is used or intended for use in combination with one or more other neurodegenerative disease therapies, including but not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, or surgery. In some embodiments, the antibody composition is administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies or other agents known to those skilled in the art for the treatment a neurodegenerative disease.

[0085] Production

[0086] The anti-Siglec-10 antibody or anti-Siglec-8 antibody, or antigen binding fragment thereof, described herein may be prepared using a eukaryotic expression system. The expression system may entail expression from a vector in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The system may also be a viral vector, such as a replication-defective retroviral vector that may be used to infect eukaryotic cells. The antibodies may also be produced from a stable cell line that expresses the antibody from a vector or a portion of a vector that has been integrated into the cellular genome. The stable cell line may express the antibody from an integrated replication-defective retroviral vector.

[0087] The anti-Siglec-10 antibody or anti-Siglec-8 described herein, or antigen binding fragment thereof, may be purified using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, fusion proteins may be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, the antibodies described herein comprising the Fc region of an immunoglobulin domain may be isolated from cell culture supernatant or a cytoplasmic extract using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) may be used to aid polypeptide purification. Such tags may be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that may be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.Immunoaffinity chromatography also may be used to purify polypeptides.

[0088] Pharmaceutical compositions-17-105756061.1Attorney Docket No. 111005.0901.01PC00

[0089] Provided herein is a pharmaceutical composition comprising a therapeutically effective amount of one or more of the anti-Siglec-10 and anti-Siglec-8 compositions described herein, or combinations thereof, and a physiologically acceptable carrier or excipient. The pharmaceutical composition may comprise a prophylactically or therapeutically effective amount of the anti- Siglec-10 antibody or anti-Siglec-8 antibody, or combination thereof, and a pharmaceutically acceptable carrier.

[0090] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0091] Generally, the ingredients of the pharmaceutical composition may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

[0092] The pharmaceutical composition may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such-18-105756061.1Attorney Docket No. 111005.0901.01PC00 as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0093] Methods of administration

[0094] Methods of administering the anti-Siglec-10 antibody and anti-Siglec-8 antibody compositions and the pharmaceutical compositions thereof described herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e g., intranasal and oral routes). In a specific embodiment, the antibodies of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.EXAMPLES

[0095] The disclosure has multiple aspects, illustrated by the following non-limiting examples.EXAMPLE 1Generation of monoclonal anti-Siglec-10 antibodies

[0096] To generate an antagonist, mice were immunized with Siglec-10-transfected murine cell lines, after three immunizations, spleen cells were harvested for generation of hybridoma. The hybridoma supernatants were screened for their activity in reversing the inhibitory effect of Siglec-10 in ADCC, which was measured using effector cells expressing human FcyRIIIa. Out of more than 20,000 clones, one (“31F11”) was found to be most potent in reversing the inhibition by Siglec-10 (Fig. 1A). Compared with another anti-Siglec-10 antibodies (10H3 and 5G6), 31F11 was more potent in antagonizing the Siglec-10 function.

[0097] To confirm the specificity of 31F11, plates were plated with fusion proteins consisting of the extracellular domains of Siglecs 1, 2, 3, 5, 6, 7, 9, 10, and 11 and the binding of 31F11 to these Siglecs was tested. As shown in Figure IB, only Siglec-10 showed appreciable binding to 31F11. As an alternative approach, we transfected 293T cell line with GFP -tagged Siglecs 2, 3,-19-105756061.1Attorney Docket No. 111005.0901.01PC005, 9, 10, 12, and 15. As shown in Figure 1 C, 31F1 1 binds specifically to cells expressing Siglec- 10, but not any other Siglecs tested. Taken together, data in Figures IB and 1C demonstrate that 31F11 binds to Siglec-10, but not Siglecs 1, 2, 3, 5, 6, 7, 9, 11, 12 and 15.EXAMPLE 2Humanization of 31F11

[0098] By blasting a human Ig database, human germline V region sequence IGHV2-70*04 and J region sequence JH6 were applied as the human framework acceptor for the CDR regions of 31F11 VH. Human germline V region IGKV3-15*01 and J region sequence JK4 were applied as the human framework acceptor for the CDR regions of 31F11 VL. 5 huVH versions (VHvl, VHv2, VHv3, VHv4, and VHv5, having SEQ ID NOs: 9-13) and 5 huVL versions (VLvl, VLv2, VLv3, VLv4, and VLv5, having SEQ ID NOs: 14-18) were designed.

[0099] To select the best working combination(s) of HuVH and HuVL for Siglec-10 binding, DNA encoding the heavy and light chains were synthesized in expression constructs and different combinations were co-tansfected into 293 cells, 7 humanized antibodies and the chimeric parental antibody were compared for their binding to Jurkat cells expressing human Siglec-10. The properties of the clones are summarized in Table 1.Table 1 Characterization humanized clones of 3 IF 11-20-105756061.1Attorney Docket No. 111005.0901.01PC00

[0100] The antibody combinations are as follows: #21 (SEQ ID NOS: 9 and 15), #22 (SEQ ID NOS: 10 and 15), #23 (SEQ ID NOS: 11 and 15), #32 (SEQ ID NOS: 10 and 17), #34 (SEQ ID NOS: 12 and 17), #52 (SEQ ID NOS: 10 and 16), and #54 (SEQ ID NOS: 12 and 16). Among them, the parental antibody had an ECso of 210 ng / ml, while the humanized antibodies had an ECso between 228 ng / ml and 423 ng / ml. This suggests that all antibodies showed potent binding to cell surface Siglec-10. Thermal stability analysis showed that all humanized antibodies exhibit good thermal stability. Using Biacore analysis, we measured the affinity of the 5 top ranked antibodies. As shown in Table 1, all 5 antibodies tested show high affinity binding for Siglec-10. In particular, three of the five antibodies (#22, #23 and #52) had higher affinity than the chimeric parental clone.EXAMPLE 3 Role of Siglec-8 and Siglec-10 in pathogenesis of AD

[0100] Analysis of single-cell RNA sequencing data from Allen Brain Cell Atlas revealed that Siglec-10 and Siglec-8 are most abundantly and almost exclusively expressed in the human microglia, while Siglec-12 is not expressed in the brain (Figure 2A). To evaluate the function of these Siglec, we developed a transgenic mouse model using a genomic clone comprising of human Siglec-8, 10 and 12 genes. Use of the genomic clone with human regulatory and coding sequences may allow the mouse to express Siglec-8 and Siglec-10 in a manner substantially similar to that in human brain cells. Flow cytometry confirmed that expression of Siglec-8 and Siglec-10 genes in the brain cells of the Siglec transgenic line (Siglec TG mice, also called “TG” mice). As shown in Figure 2B, in the brain single-cell suspension prepared from the Siglec TG mice, Siglec-8 and Siglec-10 are expressed at high levels in >90% of microglia (as identified by the expression of CDl lb and CX3R1 markers). In contrast, <1% of other brain cells expressed Siglec-8 and Siglec-10 proteins. These data suggest that the Siglec TG mice largely recapitulate expression of human Siglec-8 and Siglec-10 transgene in the brain.

[0101] A recent report showed that expression of Siglec-10 is elevated in AD patients when compared with age-matched controls. To test if human Siglec-8 and Siglec-10 genes affect the AD pathogenesis, we compared accumulation of Ab amyloid in the brain of age-matched TG and WT housed in the same facility. As shown in Figure 3A, the brain section of the TG mice exhibited larger and higher number of amyloid plaques than the WT. Quantitative image analysis-21-105756061.1Attorney Docket No. 111005.0901.01PC00 showed the increases in both plaque number of intensity (Figure 3B). We also evaluated pTau accumulation in the two groups of mice. As shown in Figure 3C &3B, higher level of pTau accumulation was observed in both cortex and hippocampus of TG mice when compared with WT mice. These data demonstrate that human Siglec-10 and / or Siglec-8 protein expression in the microglia likely contributed to pathogenesis of AD and possibly other neurodegenerative diseases. The TG mice disclosed herein can be used for screening for therapeutics targeting these human genes for the treatment of neurodegenerative diseases, including AD.EXAMPLE 4Therapeutic targeting Siglec-10 for Alzheimer's disease by anti-Siglec-10 mAh

[0102] To evaluate the therapeutic activity of anti-Siglec-10 antibodies in the mice, we treated 9- month-old TG mice with a mouse anti-human Siglec-10 monoclonal antibody, 31F11 at 3 mg / injection, twice a week for 4 weeks and analyzed the AD-associated pathological changes at the end of 4 weeks (diagramed at the top of Figure 4A), PBS was used as control. The bar graph in Figure 4A summarized the number and intensity of Ab amyloid plaques, while the images in Figure 4B depicted Ab amyloid plaques in the cortex and hippocampus of the two groups. These data showed that 31F11 significantly reduced pathology associated with the Ab amyloid plaques. Likewise, significant reduction of pTau pathology was detected in both cortex and hippocampus (Figure 4C-E).

[0103] Transgenic mice overexpressing PSEN1*M146V and APP*Swe mutations under the control of Thyl promoter (ARTE10) is a commonly used AD model. We therefore crossed the Siglec TG mice with the Taconic ARTEIO mouse model 16347 and evaluated the therapeutic efficacy of 3 IF 11. As shown in Figure 5 A (left) diagrams, the Fl mice received intraperitoneal treatments of 1 mg / injection of 31F11 or PBS control, twice a week for 6.5 weeks and then analyzed for the Ab amyloid accumulation. Low power images showed high level of Ab amyloid accumulation in the control group, and largely absence of the 31F11 treated group. Near complete clearance is observed in the treated group is further conformed in high power images in Figure 5B. Consistent with therapeutic activities, 3 IF 11 treatment abrogated binding of 3 IF 11 binding to microglia dendrites, although some binding to the cell body was seen, perhaps due to intracellular Siglec-10 protein that are largely inaccessible to antibodies (Figure 5C).-22-105756061.1Attorney Docket No. 111005.0901.01PC00

[0104] To expand this observation, we produced SIGLECTG xPrnp-MAPT*P301L Fl mice that express both human Siglec targets controlled by their own promoter and pathogenic Tau mutation under the control of mouse prion protein promoter. As shown in Figure 6A left diagrams, the Fl mice received intraperitoneal treatments of 1 mg / inj ection of 3 IF 11 or PBS control, twice a week for 6.5 weeks and then analyzed for the total Tau and pTaul81 in the plasma. As shown in Figure 6B and 6C, significant reduction in both total Tau and pTau was observed in mice treated with the anti-Siglec 10 mAb. Notably, while the control mice show broad distribution of the Tau and pTau proteins, as expected, anti-SIGLEC 10-treated mice all have low Tau and pTau levels, which showed consistent effect of the therapeutic antibody.EXAMPLE 5Humanized anti-SigleclO antibody ONC-841 effectively saturates Siglec 10 in the CNS

[0105] Since humanized 31F11 (“ONC-841 ”) induces significant anti-drug antibodies (“ADA”) that prevented chronic dosing, we tested if the humanized antibody is capable of penetrating into CNS. As shown in Figure 7A, the Siglec TG mice received intravenous injections of ONC-841 at 200pg / mouse or 400pg / mouse, roughly translated into 10 mg / kg or 20 mg / kg based on the mouse body weight of 20 gram, Qldx5. After perfusion to remove circulating antibodies, the mouse brains were harvested and analyzed by flow cytometry and immunohistochemistry for the un-occupied Siglec- 10 protein on the cell surface. As shown in Figure 7B, gated microglia in the single cell suspension from the PBS treated mice all showed strong binding to ONC-841, while those that received 20 mg / kg of ONC-841 treated mice show essentially no binding to ONC-841. At 10 mg / kg, the binding is reduced by more than 90% (Figure 7C). The percentage of microglia in ONC-841 treated mice are comparable to the PBS-treated mice (Figure 7B). Consistent with the flow cytometry data, immunohistochemistry staining with anti-Siglec- 10 monoclonal antibody showed unoccupied Siglec- 10 on the cell surface (including dendrites) are largely absent at either dose, although staining inside the cells remains largely intact (Figure 7D). The number of microglia was not affected by ONC-841 (Figure 7E). These data demonstrated that after systemic treatment, ONC-841 is capable of reaching the CNS and blocking Siglec-10 function at therapeutically feasible doses.-23-105756061.1Attorney Docket No. 111005.0901.01PC00EXAMPLE 6Anti-Siglec 10 antibody promotes phagocytosis of pathogenic fibrins by microglia

[0106] To test the hypothesis that anti-Siglec-10 mAh ameliorates AD pathology by promoting phagocytosis of pathogenic fibrins in the CNS, we isolated single cell suspension from the Siglec TG mice and pretreated them with various doses on ONC-841 and then analyzed phagocytosis of the fibrin by flowcytometry (Figure 8A, Figure 9 A). An example of Ab fibrin phagocytosis is shown in Figure 8B, which indicate a 2-fold increase in phagocytosis by microglia when 2 pg / ml of ONC-841 was used. The enhancement is dose-dependent with an EC50 of 0.127 pg / ml. Similar enhancement of Tau PFF by ONC-841 (Figure 9) was also seen. To evaluate the function of ONC-841 on human microglia, we generated microglia from human iPSC and incubated them with ONC-841 or PBS for 30 minutes before testing the phagocytosis of AD-related Tau PFF. As shown in Figure 10, ONC-841 effectively enhanced phagocytosis of Tau PFF, with an EC50 of 0.007 pg / ml. The effective enhancement of microglia phagocytosis of pathogenic fibrins not only provided a mechanism for the therapeutic activities of ONC-841, but also provided an in vitro assay to screen for other therapeutic anti-Siglec-10 antibodies.EXAMPLE 7Anti-Siglec-8 antibody promotes phagocytosis of pathogenic fibrins by microglia

[0107] To test if anti-Siglec-8 monoclonal antibody promote phagocytosis of pathogenic fibrins by microglia, we isolated single cell suspension from the Siglec TG mice and pretreated them with various doses on ONC-941m. ONC-941m is a mouse anti-Siglec 8 mAh produced by OncoC4, Inc., based on the protein sequence of a known anti-Siglec 8 mAb. As shown in Figure 11 and Figure 12, ONC-941m promoted phagocytosis of the fibrin, as determined by flowcytometry (Figure 11A, Figure 12A). An example of A0 fibrin phagocytosis is shown in Figure 1 IB, which indicates a 2-fold phagocytosis by microglia when 2 pg / ml of ONC-941m was used. The enhancement is dose-dependent with an EC50 of 0.047 pg / ml. To evaluate the function of ONC-941m on human microglia, we generated microglia from human iPSC and incubated them with ONC-941m or PBS for 30 minutes before testing the phagocytosis of AD- related Tau PFF. As shown in Figure 11, ONC-941m effectively enhanced phagocytosis of Tau PFF, with an EC50 of 0.008 pg / ml. The effective enhancement of microglia phagocytosis of pathogenic fibrins suggests that Siglec-8 is a therapeutic target for AD or other neurological-24-105756061.1Attorney Docket No. 111005.0901.01PC00 diseases. The method of in vivo testing disclosed herein will enable others to develop anti-Siglec 8 monoclonal antibodies for treatment of AD patients.

[0108] Humanization of ONC-941m

[0109] To develop ONC-941m for clinical use, we designed humanized ONC-941m heavy and light chain variable regions. As a basic strategy, we grafted CDRs from ONC-941m into closest human variable regions. In some designs, we also modified CDR region to identify more suitable antibodies. The designed sequences are provided in the table below. For the constant region, we used a human IgG4 variant with a S266P substitution (position relative to the heavy chain sequence of ONC-941m) and half-life extending LS substitutions (SEQ ID NO: 36).

[0110] To identify clones with highest affinity, one may pair different light chains and heavy chains to generate a large panel of different mAb. The affinities of these mAbs for human Siglec- 8 will be compared with that of the ONC-941m. Functional characterization of the antibodies will be performed using examples provided herein. The humanized antibodies to be chosen for clinical development should have better humanization scores, comparable to ONC-941m in affinity toward Siglec 8, penetration to the brain, enhance phagocytosis of pathogenic fibrins, and ameliorate pathology and behavior defects in AD models.Table 2 CDR sequences of humanized ONC-941mEXAMPLE 8Therapeutic effects of 31F11 in aged SigleclO / ARTE mice[0U1] This example demonstrates that an antibody disclosed herein has therapeutic effects in aged mice. To test the therapeutic effect of 31F11 on reducing AD pathology on aged mice, 13 month old siglecg KO / SigleclO ARTE Fl mice were treated with either PBS or 31F11,-25-105756061.1Attorney Docket No. 111005.0901.01PC001 mg / mouse / inj ection, TP, twice a week for 14 injections. Mice were euthanized at 15 months of age. Brain sections were fixed and stained with anti-human amyloid beta antibody6E10. Representative brain sections showed the amyloid beta plaque staining from neocortex and hippocampus area from PBS- and 31F11-treated mice (FIG. 13). Amyloid plaques were quantified. FIG. 14 shows the average amyloid load (percentage of the whole area) and the average plaque number in each area. The results demonstrate that 3 IF 11 treatment significantly reduced amyloid beta pathology after chronic treatment.

[0112] The effects on astrogliosis were also tested. Astrogliosis is a process in the nervous system where astrocytes, a type of glial cell, become activated and proliferate in response to injury, disease, or other stimuli. It is essentially a reactive response by astrocytes, characterized by changes in their morphology, number, and function. The astrocyte marker GFAP is usually used to assess astrogliosis. Astrogliosis was evaluated using anti-GFAP antibody (EPR1034Y) on brain sections from 13 month old siglecg KO / SigleclO / ARTE Fl mice treated with either PBS or 31F11, 1 mg / mouse / inj ection, intraperitoneally, twice a week for 14 injections. Astrocyte intensity (FIG. 16) and abundance (FIG. 15) were significantly reduced following 31F11 treatment compared with PBS control, indicting a more favorable brain environment after anti- Siglecl 0 therapy.

[0113] Further, 31F11 treatment changed microglia morphology toward normal status. Microglia morphology changes in response to disease conditions, exhibiting a spectrum of shapes from ramified to amoeboid. These morphological changes are linked to various neurological disorders, including Alzheimer's disease. Ramified microglia are characterized by a small cell body and numerous thin, branching processes. These microglia are in a surveillance state, constantly monitoring their environment and clear culprits in central nervous system. In response to injury or disease, microglia can transform into an activated state, characterized by changes in morphology and function. Amoeboid morphologies are widely observed in disease associated microglia, with a large, rounded cell body and short, reduced processes. By using microglia marker IBA1 staining, we thoroughly quantified the microglial morphology changes following 31F11 chronic treatment. The results are shown in FIG. 17.

[0114] In AD, the thickness of pyramidal neuron layers (both thinning and changes in neuronal density), particularly in the hippocampus and neocortex, can be affected by amyloid pathology. The neuronal marker NeuN staining in hippocampus CAI area is widely used to-26-105756061.1Attorney Docket No. 111005.0901.01PC00 indicate the neurodegeneration associated with amyloid beta pathology. Brain sections from 13 month old siglecg KO / SigleclO / ARTE F 1 mice treated with either PBS or 3 IF 11,1 mg / mouse / inj ection, IP, twice a week for 14 injections were observed. FIG. 18 shows that 31F11 treatment restored neurodegeneration observed in hippocampus pyramidal layer. The data show that both thickness of the pyramidal layer and neuron density increased upon treatment.

[0115] It was next determined whether the enhanced amyloid burden clearance is manifested in behavior changes. Anxiety-like behaviors were evaluated using the elevated plus maze (EPM). In the EPM, rodents are presented with a choice between exploring open and enclosed arms, and their behavior provides insights into their anxiety levels (FIG. 19A). For AD mouse models, the EPM can reveal impairments in emotional or cognitive domains related to the disease. Rodents naturally prefer enclosed spaces due to a fear of open, exposed areas. In contrast, AD mice with pathology development show increased time spent in open arms. As shown in FIG. 19B, 31F11 treated mice spent considerably less time in the open arms compared to the PBS treated mice. Moreover, when compared to the EPM score tested before the treatment, 3 IF 11 treated group showed significant improvement compared to the control group (FIG. 19C). These results indicate that 31F11 rescued the fear of heights and open spaces and mitigates risk-taking behavior in AD mice.

[0116] The cognitive function of mice in learning and memory was evaluated using the novel objective recognition test (NORT). NORT is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The NORT can be completed over 3 days: a habituation day, a training day, and a testing day. During training, the mouse is allowed to explore 2 identical objects. On the test day, one of the training objects is replaced with a novel object (FIG. 20A). Because mice have an innate preference for novelty, if a mouse recognizes the familiar object, it will spend most of its time at the novel object. In model AD mice, due to cognition impairment, mice cannot distinguish the novel object from a familiar one. As shown in FIG. 20B, 31F11 treated mice display increased preference for the novel object, indicating improved memory related functions following treatment. Collectively, anti- SigleclO treatment in AD model mice improved memory and cognitive behaviors.

[0117] To carry out treatment with multiple doses of 3 IF 11 (ONC-841), 15-month-old SIGLEC10;ARTE10 mice were given AI-0216 (an antibody-drug conjugate (ADC) towards B cells) prophylactically at 5 mg / injection / mouse on Day -12, -9 and -7 to minimize cross--27-105756061.1Attorney Docket No. 111005.0901.01PC00 species hypersensitivity reactions. Mice were then treated with ONC-841 via intraperitoneal injections at 20 mg / kg or PBS vehicle control for 8 weeks. Representative images of brain sections stained for A show a reduction of Ap in the ONC-841 -treated mice compared to the PBS control mice (FIG. 21A). The quantification (FIG. 21B) shows significant decrease of average plaque area and a tendency of reduced plaque numbers following anti-SigleclO treatment. This finding is consistent with the anti-sigleclO (31F11) treatment data discussed above. Together these data suggest that treatment with anti-SIGLEClO induced clearance of A plaques, thereby abrogating pathological changes in a mouse model of Alzheimer’s disease.

[0118] By staining with the microglia marker IBA1, microglial morphology changes following ONC-841 chronic treatment were quantified. The results are shown in FIG. 22A-B. In addition, ONC-841 treatment significantly reduced astrogliosis, as shown in FIG. 23A-B.105756061.1

Claims

Attorney Docket No. 111005.0901.01PC00CLAIMS1. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering an anti-Siglec-10 antibody to the subject.

2. The method of claim 1, wherein the neurodegenerative disease is selected from Alzheimer’s diseases, dementia, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease.

3. The method of claim 2, wherein the neurodegenerative disease is Alzheimer's disease.

4. The method of claim 1, wherein the anti-Siglec-10 antibody ameliorates the accumulation of A0 amyloid in the brain.

5. The method of claim 1, wherein the anti-Siglec-10 antibody ameliorates the accumulation of phosphorylated Tau protein in the brain.

6. The method of claim 1, wherein the anti-Siglec-10 antibody further enhances phagocytosis of amyloid or phosphorylated Tau fibrins by microglia.

7. The method of claim 1 , wherein the anti-Siglec-10 antibody penetrates CNS and occupies Siglec-10 receptor on the microglia.

8. The method of claim 1, wherein the anti-Siglec-10 antibody comprises (a) a heavy chain variable region comprising one or more of a complementarity determining region (CDR) 1 comprising the sequence set forth in SEQ ID NO: 3, a CDR2 comprising the sequence set forth in SEQ ID NO: 4, and a CDR3 comprising the sequence set forth in SEQ ID NO: 5; and, (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 6, a CDR2 comprising the sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the sequence set forth in SEQ ID NO: 8.

9. The method of claim 8, wherein the anti-Siglec-10 antibody comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 2.

10. The method of claim 9, wherein the anti-Siglec-10 antibody is 31F11.-29-105756061.1Attorney Docket No. 111005.0901.01PC0011 . The method of claim 8, wherein the heavy chain variable region comprises the sequence set forth in one of SEQ ID NOS: 9-13, and the light chain variable region comprises the sequence set forth in one of SEQ ID NOS: 14-18.

12. The method of claim 11, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 9, 10, or 11, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 15.

13. The method of claim 12, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 9.

14. The method of claim 13, wherein the anti-Siglec-10 antibody comprises a heavy chain comprising the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 9 and a constant region comprising a human IgG4.

15. The method of claim 14, wherein the human IgG4 comprises the sequence set forth in SEQ ID NO: 21.

16. The method of claim 1, wherein the anti-Siglec-10 antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 15.

17. The method of claim 16, wherein the anti-Siglec-10 antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 22 and a light chain comprising the sequence set forth in SEQ ID NO: 23.

18. The method of claim 17, wherein the anti-Siglec-10 antibody is ONC-841.

19. The method of claim 12, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 10.

20. The method of claim 12, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 11 .

21. The method of claim 11, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 10 or 12, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 17.-30-105756061.1Attorney Docket No. 111005.0901.01PC0022. The method of claim 21, wherein the heavy chain variable region comprises the sequence set forth in SEQ NO: 10.

23. The method of claim 21, wherein the heavy chain variable region comprises the sequence set forth in SEQ NO: 12.

24. The method of claim 11, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 10 or 12, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16.

25. The method of claim 24, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 10.

26. The method of claim 24, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 12.

27. The method of claim 1, wherein the anti-Siglec-10 antibody is a bi-specific antibody, and wherein the anti-Siglec-10 antibody further comprises an anti-Siglec-8 antibody domain.

28. The method of claim 27, wherein the anti-Siglec-8 antibody domain comprises (a) a heavy chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 26 or 27; a CDR2 comprising the sequence set forth in SEQ ID NO: 28 or 29; and a CDR3 comprising the sequence set forth in SEQ ID NO: 30; and (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 31 or 32; a CDR2 comprising the sequence set forth in SEQ ID NO: 33; and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or 35.

29. The method of claim 28, wherein the anti-Siglec-8 antibody domain comprises the heavy chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 26, and the CDR2 comprising the sequence set forth in SEQ ID NO: 28; and the light chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 31, and the CDR3 comprising the sequence set forth in SEQ ID NO: 34.

30. The method of claim 28, wherein the anti-Siglec-8 antibody domain comprises a heavy chain comprising the heavy chain variable region and a human IgG4 constant region comprising the sequence set forth in SEQ ID NO: 36.-31-105756061.1Attorney Docket No. 111005.0901.01PC0031 . The method of claim 27, wherein the anti-Siglec-8 antibody domain comprises Lirentelimab, 7C9, or an antibody binding fragment thereof.

32. A method of treating a neurodegen erative disease in a subject in need thereof, comprising administering an anti-Siglec-8 antibody to the subject.

33. The method of claim 32, wherein the neurodegenerative disease is selected from Alzheimer’s diseases, dementia, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt- Jakob disease.

34. The method of claim 33, wherein the neurodegenerative disease is Alzheimer’s disease.

35. The method of claim 32, wherein the anti-Siglec-8 antibody ameliorates the accumulation of A0 amyloid in the brain.

36. The method of claim 32, wherein the anti-Siglec-8 antibody ameliorates the accumulation of phosphorylated Tau protein in the brain.

37. The method of claim 32, wherein the anti-Siglec-8 antibody enhances phagocytosis of amyloid or phosphorylated Tau fibrins by microglia.

38. The method of claim 32, wherein the anti-Siglec 8 antibody penetrates CNS and occupies Siglec-8 on the microglia.

39. The method of claim 32, wherein the anti-Siglec-8 antibody comprises (a) a heavy chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 26 or 27; a CDR2 comprising the sequence set forth in SEQ ID NO: 28 or 29; and a CDR3 comprising the sequence set forth in SEQ ID NO: 30; and (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 31 or 32; a CDR2 comprising the sequence set forth in SEQ ID NO: 33; and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or 35.

40. The method of claim 39, wherein the anti-Siglec-8 antibody comprises the heavy chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 26, and the CDR2 comprising the sequence set forth in SEQ ID NO: 28; and the light chain-32-105756061.1Attorney Docket No. 111005.0901.01PC00 variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 31, and the CDR3 comprising the sequence set forth in SEQ ID NO: 34.

41. The method of claim 39, wherein the anti-Siglec-8 antibody comprises a heavy chain comprising the heavy chain variable region and a human IgG4 constant region comprising the sequence set forth in SEQ ID NO: 36.

42. An anti-Siglec-8 antibody or antigen binding fragment thereof comprising: (a) a heavy chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 26 or 27; a CDR2 comprising the sequence set forth in SEQ ID NO: 28 or 29; and a CDR3 comprising the sequence set forth in SEQ ID NO: 30; and (b) a light chain variable region comprising one or more of a CDR1 comprising the sequence set forth in SEQ ID NO: 31 or 32; a CDR2 comprising the sequence set forth in SEQ ID NO: 33; and a CDR3 comprising the sequence set forth in SEQ ID NO: 34 or 35.

43. The anti-Siglec-8 antibody or antigen binding fragment thereof of claim 42, comprising the heavy chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 26, and the CDR2 comprising the sequence set forth in SEQ ID NO: 28; and the light chain variable region comprising the CDR1 comprising the sequence set forth in SEQ ID NO: 31, and the CDR3 comprising the sequence set forth in SEQ ID NO: 34.

44. The anti-Siglec-8 antibody or antigen binding fragment thereof of claim 42, comprising a heavy chain comprising the heavy chain variable region and a human IgG4 constant region comprising the sequence set forth in SEQ ID NO: 36.

45. A pharmaceutical composition comprising the anti-Siglec-8 antibody of claim 42 and a pharmaceutically acceptable carrier or excipient.

46. The anti-Siglec-8 antibody of claim 42 for use in treatment.

47. A method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-10 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.-33-105756061.1Attorney Docket No. 111005.0901.01PC0048. A method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-8 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.

49. A method of identifying a neurodegenerative disease related therapeutic agent, comprising contacting a mouse expressing Siglec-10 and Siglec-8 predominantly in the microglia with a candidate agent, and measuring the amount of phagocytosis of pathogenic fibrins by the microglia, wherein an increase in the amount of phagocytosis is indicative that the candidate agent is a neurodegenerative disease related therapeutic agent.

50. The method of any one of claims 47-49, where the mouse comprises a genomic clone of human Siglec genes, comprising at least Siglec-8 and Siglec-10 coding and regulatory sequences.-34-105756061.1

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