WO2018154580A1 - Fc-based polypeptides and use thereof - Google Patents

Abstract

The present invention is directed to methods comprising administration of immunoglobulin derived Fc fragments to a subject afflicted with a neurodegenerative disease. Further provided are compositions and methods for enhancing microglia phagocytosis using said immunoglobulin derived Fc fragments.

Description

FC-BASED POLYPEPTIDES AND USE THEREOF

CROSS REFERENCE

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/462,965 filed February 24, 2017, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention relates to immunoglobulin derived Fc fragments and their use in treatment of neurological diseases.

BACKGROUND OF THE INVENTION [003] Neurodegenerative diseases are generally characterized by the loss of neurons from one or more regions of the central nervous system. They are complex in both origin and progression and have proved to be some of the most difficult types of disease to treat. In fact, for some neurodegenerative diseases, there are no drugs available that provide significant therapeutic benefit. The difficulty in providing therapy is all the more tragic given the devastating effects these diseases have on their victims.

SUMMARY OF THE INVENTION

[004] The present invention relates to methods and compositions for treating neurological diseases in a subject in need thereof. In some embodiments, there is provided use or administration of immunoglobulin derived Fc fragments for treatment of neurological diseases. In some embodiments, the present invention provides a method for enhancing phagocytic activity of microglia.

[005] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[006] According to one aspect, there is provided method for enhancing phagocytic activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunoglobulin Fc fragment, wherein the Fc fragment is characterized by an increased binding affinity to an Fc receptor, thereby enhancing phagocytic activity of the microglia cells in the subject.

[007] According to another aspect, there is provide a method for treating a neuronal disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunoglobulin Fc fragment, wherein the Fc fragment is characterized by increased binding affinity to an Fc receptor on microglia cells, thereby treating the neuronal disorder in the subject.

[008] In some embodiments, the Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or derivative, a fragment or an analog thereof. In some embodiments, the Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof. In some embodiments, the Fc fragment comprises two polypeptides, each polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1. [009] In some embodiments, the Fc fragment is an antagonist of an Fc receptor. In some embodiments, the Fc fragment comprises a glycosylated asparagine amino acid residue. In some embodiments, the glycosylated asparagine is located at amino acid position 297 of the IgG heavy chain (N297). In some embodiments, N297 comprises a bisecting N-acetyl glucosamine (GlcNAc). [010] In some embodiments, the phagocytic activity is phagocytic activity of microglia. In some embodiments, the Fc receptor is an Fc receptor expressed on microglia cells. In some embodiments, the Fc receptor is CD 16. In some embodiments, increased binding affinity is characterized by a dissociation constant (Kd) between 0.1-10 micro molar (μΜ).

[011] In some embodiments, the neuronal disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and age- related macular degeneration (AMD).

[012] According to another aspect, there is provided a pharmaceutical composition comprising an immunoglobulin Fc fragment and a pharmaceutically acceptable carrier, wherein the Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof, for use in enhancing microglia phagocytosis. [013] According to another aspect, there is provided a pharmaceutical composition comprising an immunoglobulin Fc fragment and a pharmaceutically acceptable carrier, wherein the Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof, for use in treating a neurodegenerative disease selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and age-related macular degeneration (AMD).

[014] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[015] Figs. 1A-1L are representative confocal microscopic images of brain (A-F) and SC (G-L) taken from 136-day-old mSODl^G93A mice injected with either Fc-rituximab or PBS and immunohistochemically stained specifically for nuclei (DAPI; blue) and a neuronal nuclear antigen (NeuN+; green). Scale bar- 100 μιη.

[016] Fig. 2 shows distribution of the Fc-rituximab in mice brains 2.5 hours following injection of labeled Fc-rituximab with Alexa Fluor 680. Brains from mSODl^G93Amice showed a higher signal intensity in comparison to WT mice brains in both male and female.

[017] Figs. 3A-3B are bar graphs showing TNF-alpha levels, as measured by ELISA, in cultured primary microglia cells obtained from brain (A) and SC (B) of mSODl^G93A and wild type mice treated with Fc-rituximab or PBS and sacrificed at 120 days old (n=6).

[018] Fig. 4 is a bar graph showing TNF-a release in cultured primary microglial cells obtained from brains of sacrificed mSODl^G93A mice following various treatments (fetal bovine serum (Blank), Fc-Rituximab (0.12 mg/ml), intact Rituximab (1 mg/ml) or LPS).

[019] Figs. 5A-5D are bar graphs showing CCL2 (A, B) and IL-l-beta (C, D) levels in microglia cells isolated from brains. (B, D) and SC (A, C) of both mSODl and WT mice 7 days after injection of Fc-rituximab or PBS. All samples were normalized to GAPDH housekeeping gene and to mSODl mice injected with PBS. [020] Figs. 6A-6D demonstrate phagocytosis of apoptotic NSC34 cells by microglia cell line (BV-2 cells) incubated for 16 h in RPMI medium supplemented with inactivated fetal bovine serum (Blank) (A), Fc-rituximab (0.116 mg/ml) (B) or intact rituximab (0.25-1 mg/ml) (C). Phagocytic index was calculated for cells presented in A-C (D). [021] Figs. 7A-7D demonstrate Fc scatters, binds to microglia and keeps the number of motor neurons in the CNS of S0D1^G93A mice. Live imaging of Fc conjugated with AF 680 shows scattering of the Fc in the brain and spinal cord tissues up to 4 h, and clearance after 8h (A). Representative immunofluorescent image depicts co-localization (pointed by arrows) of the Fc with microglia in brain of SODl⁰⁹^ male mice 24h after injections (B), Iba+ and CD1 lb+ cells isolated from spinal cords of S0D1^G93A mice bind Fc and intact IgG (Rituximab) (C) and representative immunofluorescent image depicts neurons (NeuN+; green) co-stained with nuclear staining (DAPI; blue) in lumbar level of spinal cords harvested from SODl⁰⁹^ males 50 days after Fc and PBS administration (D). Data are mean + s.e.m.

[022] Figs. 8A-8E demonstrate one intrathecal injection with Fc extends lifespan, improves motor functions, and slows the rate of disease progression in S0D1^G93A males. Kaplan-Meier analysis of the probability of surviving as a function of age in Fc- and PBS- injected S0D1^G93A mice (n=17 males, n=12 females, n=15 littermates). Mice were once injected by Fc or PBS at day 75. Results of Fc injection show prolonged survival by 21 days (p<0.0065) relative to PBS in males (A), weight loss after symptom onset and gait impairment score of S0D1^G93A mice became worse with age, but this was attenuated by the Fc treatment for S0D1^G93A males (B), insignificant changes (p<0.5) in lifespan of females (C), weight loss was performed when female foot was dragging and differences in neurological score of S0D1^G93A females were seemed at limb paralysis (D). Lifespan of injected littermates was unaffected (E). Neurological score: no symptoms =0, cannot right itself within 30 s after being placed on either side =5. Data are mean + s.e.m. *P<0.05, **P<0.01, ***P<0.001.

[023] Figs. 9A-9D are vertical bar charts describing gene expression of cytokines and proteins in spinal cord tissues of S0D1^G93A mice on day 7 to Fc injection. Quantitative real time PCR analysis of anti-inflammatory cytokines and marker; IL-10, TGFP and arginase-1 (M2 microglia) (A), pro-inflammatory cytokines and marker; IL-Ιβ, TNFa, iNOS (Ml microglia) and CCL2 from whole spinal cords of S0D1^G93A male mice (B), IL-10, TGFP and arginase-1 (C), and IL-Ιβ, TNFa, iNOS and CCL2 from whole spinal cords of SOD 1^G93A female mice (D), (n=5 from each group) 7 days after Fc or PBS injection. Data are mean + s.e.m. *P<0.05, NS; nonsignificant. [024] Figs. 10A-10D are vertical bar charts describing gene expression of cytokines and proteins in spinal cord tissues of wild-type (WT) littermates on day 7 to Fc injection. Quantitative real time PCR analysis of anti -inflammatory cytokines and marker; IL-10, TGFP and arginase-1 (M2 microglia) (A), pro-inflammatory cytokines and marker; IL-Ιβ, TNFa, iNOS (Ml microglia) and CCL2 from whole spinal cords of WT male littermates (B), IL-10, TGFp and arginase-1 (C), and IL-Ιβ, TNFa, iNOS and CCL2 from whole spinal cords of WT female littermates (D), (n=5 from each group) 7 days after Fc or PBS injections. Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001.

[025] Figs. 11A-11F describe TNFa secretion and phagocytosis by isolated or immortalized microglia. Secretion of TNFa produced by LPS-stimulated microglia isolated from brain and spinal cord of S0D1^G93A male mice or their littermates 50 days after Fc or PBS injection (A), by Fc- and Rituximab -treated or untreated microglia of spinal cords isolated from 95 days old S0D1^G93A male mice (B), uptake of fluorescent micro-beads or UV-apoptotic NSC-34 cells by immortalized BV2 cells (C), the rate of bead uptake by untreated (Cnt), Fc- and Rituximab-treated (RtmAb) BV2 cells during 15 min (D), uptake of fluorescent micro- beads by Fc-treated or untreated primary microglia isolated from spinal cords of WT mice (E), and flow cytometry analysis of the number of beads uptake by Fc-treated or untreated primary microglia isolated from spinal cords of WT mice (F). Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001. [026] Figs 12A-12B are vertical bar charts describing gene expression of cytokines and proteins in immortalized BV2 cells. Quantitative real time PCR analysis of cytokines and marker characterizing M2 microglia; IL-10, TGFP, arginase-1 and IL-4 (A), cytokines and marker characterizing Ml microglia; IL-Ιβ, TNFa, iNOS and CCL2 (B) from Fc- and Rituximab-treated or untreated BV2 cells. Data are mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001.

DETAILED DESCRIPTION OF THE INVENTION

[027] The present invention relates to methods and compositions for treating neurological diseases in a subject in need thereof. In some embodiments, there is provided use or administration of immunoglobulin derived Fc fragments for treatment of neurological diseases. In some embodiments, the present invention provides a method for enhancing phagocytic activity of microglia.

[028] In some embodiments, the method comprises administering to a subject in need thereof an isolated antibody Fc fragment, as described herein. In some embodiments, said Fc fragment is an antagonist of CD 16. In some embodiments, the CD 16 Fc receptor is selected from: FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b).

[029] As exemplified in the example section, and without wishing to be bound by any theory or mechanism of action, the Fc fragments of rituximab increases microglia phagocytosis. As further exemplified herein, contacting microglial cells with Fc fragments of rituximab resulted in increased release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha) and chemokine (C-C motif) ligand 2 (CCL2).

Fc fragments

[030] The terms "Fc fragment" or "immunoglobulin Fc fragment" as used herein, refer to the C-terminal region of an immunoglobulin. The Fc fragment is a dimeric molecule comprising at least two disulfide-linked antibody heavy chain Fc fragment polypeptides. Fc fragments contains the heavy-chain constant region 2 (CH2) and the heavy-chain constant region (CH3) of an immunoglobulin, and not the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CHI) and the light-chain constant region 1 (CLl) of the immunoglobulin. It may further include the hinge region at the heavy-chain constant region. Also, the immunoglobulin Fc fragment of the present invention may contain a portion or all the heavy-chain constant region 1 (CHI) and/or the light-chain constant region 1 (CLl), except for the variable regions of the heavy and light chains. Also, as long as it has a physiological function substantially similar to or better than the native protein IgG Fc fragment may be a fragment having a deletion in a relatively long portion of the amino acid sequence of CH2 and/or CH3. That is, the immunoglobulin Fc fragment of the present invention may comprise 1) a CHI domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CHI domain and a CH2 domain, 3) a CHI domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer of each domain of the heavy-chain constant regions and the light-chain constant region. It should be appreciated to one skilled in the art that the immunoglobulin Fc fragment of the invention are devoid of a Fab region.

[031] In some embodiments, the Fc fragment of the present invention comprises two heavy chain polypeptides linked by at least two disulfide bonds. In another embodiment, the Fc fragment of the present invention comprises two heavy chain polypeptides linked by at between 2 to 4 disulfide bonds. In another embodiment, the Fc fragment of the present invention comprises two heavy chain polypeptides linked by at between 4 to 11 disulfide bonds. In another embodiment, the Fc fragment of the present invention comprises two heavy chain polypeptides linked by at between 11 to 20 disulfide bonds.

[032] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. In another embodiment, the hinge region of the Fc fragment of the present invention has a length of at least 12 amino acids. In another embodiment, the hinge region of the Fc fragment of the present invention has a length of at least 15 amino acids. In another embodiment, the hinge region of the Fc fragment of the present invention has a length of between 12-62 amino acids. In another embodiment, the hinge region of the Fc fragment of the present invention has a length of between 15-62 amino acids.

[033] In some embodiments, Fc fragments may be obtained from native immunoglobulins by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native immunoglobulin into Fab and Fc fragments, and pepsin treatment results in the production of pF'c and F (ab') 2 fragments. These fragments may be subjected, for example, to size exclusion chromatography to isolate Fc or pF'c.

[034] In another embodiment, a human-derived Fc fragment is a recombinant immunoglobulin Fc fragment that is obtained from a microorganism. Fc glycoform modification

[035] In another embodiment, the Fc fragment of the present invention may be subjected to glycoform modification. Many polypeptides, including antibodies and Fc fragments, are subjected to a variety of post-translational modifications involving carbohydrate moieties, such as glycosylation with oligosaccharides. There are several factors that can influence glycosylation. The species, tissue and cell type have all been shown to be important in the way that glycosylation occurs. In addition, the immunoglobulin Fc fragment of the present invention may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form or may be in a deglycosylated form. The increase, decrease or removal of the immunoglobulin Fc sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method and a genetic engineering method. [036] In another embodiment, glycoform modification of the Fc fragment of the present invention increases an effector function. In another embodiment, glycoform modification of the Fc fragment of the present invention increases binding affinity to CD16.

[037] The term "glycosylation" means the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to the Fc fragment. The oligosaccharide side chains are typically linked to the backbone of the Fc fragment through either N- or O-linkages. In some embodiments, the oligosaccharides of the present invention are attached to a CH2 domain of an Fc fragment as N-linked oligosaccharides. "N-linked glycosylation" refers to the attachment of the carbohydrate moiety to an asparagine residue in a glycoprotein chain. The skilled artisan will recognize that, for example, each of murine IgGl, IgG2a, IgG2b and IgG3 as well as human IgGl, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have at least one site for N-linked glycosylation at amino acid residue 297.

[038] As used herein, the term "residue 297" refers to the asparagine at location 301 of SEQ ID NO: 2. In some embodiments, residue 297 refers to the asparagine at location 130 of SEQ ID NO: 1. In some embodiments, said glycosylation at amino acid residue 297 is of a bisecting N-acetyl glucosamine (GlcNAc). In some embodiments, said bisecting-GlcNAc lacks a core fucose. In some embodiments, said glycosylation is A2BG2.

[039] In another embodiment, the altered glycosylation comprises an increased level of bisected complex residues in the Fc fragment.

[040] In another embodiment, the altered glycosylation comprises a reduced level of fucose residues in the Fc fragment.

[041] The term "microglia" as used herein refers to the smallest of the glial cells that can act as phagocytic cells, cleaning up CNS debris. In some embodiments, the methods and compositions of the present invention increase the secretion of pro-inflammatory cytokines from microglia. Non-limiting examples of pro-inflammatory cytokines include TNF alpha and CCL2.

Fc variants and Fc fusions

[042] In another embodiment, the CD 16 binding Fc fragment of the present invention is an Fc variant having affinity to CD 16.

[043] The term "Fc variant" as used herein refers to an Fc fragment that comprises one or more amino acid modifications relative to a WT Fc fragment, wherein the amino acid modification(s) provide one or more optimized properties. Amino acid modifications include: deletions, insertions, non-conservative or conservative substitutions or combinations thereof of one or more amino acid residues.

[044] In another embodiment, the Fc fragment of the present invention is an Fc variant having at least 90% homology to a WT Fc fragment. In another embodiment, the Fc fragment of the present invention is an Fc Variant having at least 95% homology to a WT Fc fragment. In another embodiment, the Fc fragment of the present invention is an Fc variant having at least 98% homology to a WT Fc fragment. In another embodiment, the Fc fragment of the present invention is an Fc variant having at least 99% homology to a WT Fc fragment. [045] In some embodiments, the optimized property of the Fc variant of the present invention is an increased affinity to CD 16 as compared to WT Fc fragment. By increased affinity it is meant that an Fc variant binds to an CD 16 with a significantly higher equilibrium constant of association (K_a) or lower equilibrium constant of dissociation (Kd) than WT Fc Fragment when the amounts of variant and WT Fc fragment in the binding assay are essentially the same. Accordingly, by "reduced affinity" as compared to a WT Fc fragment as used herein is meant that an Fc variant binds an Fc receptor with significantly lower K_a or higher Kd than the WT Fc fragment.

[046] In some embodiments, the Fc variant of the present invention exhibits an association constant (K_a) of CD 16 binding of at least 10 pico molar (pM). In another embodiment, the FC variant of the present invention exhibits an association constant (K_a) of CD 16 binding of at least 0.1 nano molar (nM). In another embodiment, the Fc variant of the present invention exhibits an association constant (K_a) of CD 16 binding of at least 1 nM. In another embodiment, the FC variant of the present invention exhibits an association constant (K_a) of CD 16 binding of at least 1 micro molar (μΜ). [047] In some embodiments, the optimized property of the Fc variant of the present invention is 2 to 5-fold increased affinity to CD 16 as compared to WT Fc fragment. In some embodiments, the optimized property of the Fc variant of the present invention is 5 to 10-fold increased affinity to CD 16 as compared to WT Fc fragment. In some embodiments, the optimized property of the Fc variant of the present invention is 10 to 100-fold increased affinity to CD 16 as compared to WT Fc fragment. In some embodiments, the optimized property of the Fc variant of the present invention is 100 to 1,000-fold increased affinity to CD 16 as compared to WT Fc fragment. [048] In some embodiments, the optimized property of the Fc variant of the present invention is an increased ability to activate ADCC compared to WT Fc fragment as determined by standard assays known in the art. In another embodiment, the Fc variant increases ADCC by at least 10% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases ADCC by between 10% -50% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases ADCC by between 50%- 100% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases ADCC by between 100%-500% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases ADCC by more than 500% compared to WT Fc fragment as measured by EC50 values.

[049] In some embodiments, the optimized property of the Fc variant of the present invention is an increased ability to activate CDC compared to WT Fc fragment as determined by standard assays known in the art. In another embodiment, the Fc variant increases CDC by at least 10% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases CDC by between 10%-50% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases CDC by between 50%- 100% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases CDC by between 100% -500% compared to WT Fc fragment as measured by EC50 values. In another embodiment, the Fc variant increases CDC by more than 500% compared to WT Fc fragment as measured by EC50 values.

[050] In some embodiments, the optimized property of the Fc variant of the present invention is a modification to reduce immunogenicity in humans. Modifications to reduce immunogenicity may include modifications that reduce binding of processed peptides derived from the parent sequence to MHC proteins. For example, amino acid modifications may be engineered such that there are no or a minimal number of immune epitopes that are predicted to bind, with high affinity, to any prevalent MHC alleles. Several methods of identifying MHC- binding epitopes in protein sequences are known in the art and may be used to score epitopes in an Fc variant of the present invention.

[051] In another embodiment, the optimized property of the Fc variant of the present invention is an increase in the affinity of the variant Fc fragment for FcyRIIIa (CD16A) and a decrease in the affinity of the variant Fc fragment for FcyRIIIb (CD16B), relative to a comparable molecule comprising a WT Fc fragment which binds FcyRIIIa and FcyRIIIb with WT affinity. [052] In another embodiment, the optimized property of the Fc variant of the present invention is an increase in the affinity of the variant Fc fragment for FcyRIIIb (CD16B) and a decrease in the affinity of the variant Fc fragment for FcyRIIIa (CD16A), relative to a comparable molecule comprising a WT Fc fragment which binds FcyRIIIa and FcyRIIIb with WT affinity.

[053] In another embodiment, the optimized property of the Fc variant of the present invention is increased or reduced affinity for any Fc receptor. In some embodiments, the Fc variants of the present invention are optimized to possess increased affinity for a human- activating Fc -receptors, such as, but not limited to, FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and FcyRIIIb. In another embodiment, the Fc variants are optimized to possess reduced affinity for the human inhibitory receptor FcyRIIb.

[054] In another embodiment, the alteration of affinity increases an effector function.

[055] In another embodiment, the increase in affinity or effector function is between 2- 1, 000-fold relative to a comparable molecule comprising a WT Fc fragment. In another embodiment, the increase in affinity or effector function is between 2-100-fold relative to a comparable molecule comprising a WT Fc fragment. In another embodiment, the increase in affinity or effector function is between 2-10-fold relative to a comparable molecule comprising a WT Fc fragment. In another embodiment, the increase in affinity or effector function is between 10-100-fold relative to a comparable molecule comprising a WT Fc fragment. In another embodiment, the increase in affinity or effector function is between 100-1,000-fold relative to a comparable molecule comprising a WT Fc fragment.

[056] In another embodiment, the Fc variant of the present invention is covalently modified. Covalent modifications of antibodies and antibody fragments are generally, but not always, done post-translationally. For example, several types of covalent modifications of the antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

[057] In another embodiment, the Fc variant of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.

[058] In another embodiment, the Fc fragment of the present invention is an Fc fusion. The term "Fc fusion" as used herein is a protein wherein one or more polypeptides is operably linked to an Fc fragment. An Fc fusion combines the Fc fragment of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide or small molecule. The role of the non-Fc part of an Fc fusion, i.e., the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody. Virtually any protein or small molecule may be linked to Fc fragment to generate an Fc fusion. Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such targets may be any molecule, preferably an extracellular receptor that is implicated in disease.

[059] In another embodiment, the Fc fragment of the present invention is derived from the antibody registered by ATC code L01XC02 and known as rituximab or by the commercial names RITUXAN® and MABTHERA®. This antibody is a genetically engineered chimeric human gamma 1 murine constant domain containing monoclonal antibody directed against the human CD20 antigen.

[060] In another embodiment, the Fc fragment of the present invention is isolated using a cysteine protease such as, but not limited to, papain which cleaves Fc from the Fab fragment of rituximab as described in the materials and methods section below.

[061] In another embodiment, the Fc fragment of the present invention comprises a polypeptide having at least 90%, at least 95%, at least 98% identity to the amino acid sequence set forth: HTFPA VLQS S GLYS LS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQ VYTLPPS REEMTKNQ VS LTCLVKGF YPS DIA VEWES NGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1).

[062] In another embodiment, the Fc fragment of the present invention comprises a polypeptide having at least 90%, at least 95%, at least 98% identity to the amino acid sequence set forth: Q VQLQQPG AELVKPG AS VKMS C KAS G YTFTS YNMHW VKQTPGRGLE WIG AIYPGN GDTS YNQKFKGKATLT ADKS S S T A YMQLS S LTS EDS A V Y YC ARS T Y YGGD W YFN V WGAGTT VTVX 1X2ASTKGPS VFPLAPS S KSTS GGTAALGCLVKD YFPEPVT VS WNS G ALTS GVHTFPA VLQS S GLYS LS SWT VPS S SLGTQT YICN VNHKPSNTKVDKKX3EPK

SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NN YKTTPP VLDS DGS FFLYS KLT VDKS RWQQGN VFS CS VMHE ALHNH YTQKS LS LS P GK (SEQ ID NO: 2), wherein Xi is Ser or Ala, X₂ is Ser or Ala and X₃ is Val or Ala.

[063] In other embodiments, the CD 16 binding Fc fragment of the present invention is obtained by papain cleavage of an engineered humanized anti CD20 immunoglobulin.

[064] In another embodiment, the Fc fragment of the present invention is a synthetic peptide generated by methods known in the art.

[065] In one embodiment, the peptides of the present invention, analogs or derivatives thereof produced by recombinant techniques can be purified so that the peptides will be substantially pure when administered to a subject.

[066] As used herein, the term "substantially pure" refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it. Typically, a peptide is substantially pure when at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the peptide of interest. Purity can be measured by any appropriate method, e.g., in the case of peptides by HPLC analysis. [067] In one embodiment, the peptides of the invention are peptide conjugates, comprising the peptides of the present invention derivatives or analogs thereof joined at their amino or carboxyl-terminus or at one of the side chains via a peptide bond to an amino acid sequence of a different protein. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by protein synthesis. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by use of a peptide synthesizer. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the conjugate by methods commonly known in the art. In another embodiment, addition of amino acid residues may be performed at either terminus of the peptides of the invention for the purpose of providing a "linker" by which the peptides of this invention can be conveniently bound to a carrier. In another embodiment, the linkers are comprised of at least one amino acid residue. In another embodiment, the linkers can be of 40 or more residues. In another embodiment, the linkers are comprised of 1 to 10 residues. In another embodiment, amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.

[068] The term "derivative" as used herein, refers to any polypeptide that is based off the polypeptide of the invention. A derivative is not merely a fragment of the polypeptide, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the polypeptide of the invention.

[069] As used herein, the term "derived from" or "corresponding to" refers to construction of an amino acid sequence based on the knowledge of a sequence using any one of the suitable means known to one skilled in the art, e.g. chemical synthesis in accordance with standard protocols in the art.

[070] The term "analog" as used herein, refers to a polypeptide that is similar, but not identical, to the polypeptide of the invention that still is capable of binding succinate to an Fc receptor. An analog, may have deletions or mutations that result in an amino acids sequence that is different than the amino acid sequence of the polypeptide of the invention. It should be understood, that all analogs of the polypeptide of the invention would still be capable of binding to an Fc receptor.

[071] In some embodiments, an analog to the polypeptide of the invention comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the amino acid sequence presented in SEQ ID NO: 1.

[072] As used herein, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Non-limiting examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention. [073] In another embodiment, the Fc fragment of the present invention is obtained by protease cleavage of an IgG isotype immunoglobulin. The group of IgG immunoglobulins includes the isotypes: IgGl, IgG2, IgG3 and IgG4.

[074] The term "wild-type (WT) Fc fragment" as used herein refers to an Fc fragment having the amino acid sequence identical to the amino acid sequence of an Fc fragment obtained by protease cleavage of a WT immunoglobulin found in nature.

Therapeutic methods

[075] In one aspect, the present invention provides a method of treating, delaying the onset, delaying progression of, reducing the incidence of or reducing the severity of a neuronal disease in a subject, said method comprising administering to a subject antibody derived Fc fragments or derivative thereof.

[076] In one embodiment, the neuronal disorder is a neurodegenerative disorder. In one embodiment, the neurodegenerative disorder is selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and age- related macular degeneration (AMD).

[077] In one embodiment, the neuronal disorder is a neurodegenerative disorder other than amyotrophic lateral sclerosis (ALS).

[078] In some embodiments, the term "treatment" as used herein refers to any response to, or anticipation of the neuronal disease and includes but is not limited to: preventing the neuronal disease from occurring in a subject, which may or may not be predisposed to the condition, but has not yet been diagnosed with a neuronal disease and accordingly, the treatment constitutes prophylactic treatment for a neuronal disease; inhibiting a neuronal disease, e.g., arresting, slowing or delaying the onset, development or progression of the neuronal disease; or relieving a neuronal disease, e.g., causing regression of the neuronal disease or reducing the symptoms of the neuronal disease.

[079] In another embodiment, the term "administering" as used herein, includes delivery of effective amounts of the composition of the present invention to a subject in need thereof. Methods for delivery of antibodies and antibody fragments are well known in the art.

[080] In order to treat a patient, a therapeutically effective dose of the Fc fragment of the present invention is administered. By "therapeutically effective dose" herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. In some embodiments, dosages may range from 0.01 to 1,000 mg/kg of subject body weight per day. In some embodiments, dosages may range from 0.1 to 50 mg/kg of subject body weight per day. In some embodiments, dosages may range from 1 to 100 mg/kg of subject body weight per day. In some embodiments, dosages may range from 1 to 500 mg/kg of subject body weight per day. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[081] In some embodiments, there is provided pharmaceutical compositions comprising as an active ingredient a therapeutically effective amount of the Fc fragment of the present invention, and a pharmaceutically acceptable carrier or diluents.

[082] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the Fc fragment is administered. Such pharmaceutical carriers can 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, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. 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, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

[083] The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by

E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the peptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[084] In another embodiment, administration of the pharmaceutical composition comprising the Fc fragment of the present invention, preferably in the form of a sterile aqueous solution, may be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, intraotically, transdermally, topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx® inhalable technology commercially available from Aradigm, or Inhance® pulmonary delivery system commercially available from Inhale Therapeutics), vaginally, parenterally, rectally, or intraocularly. Moreover, one may administer one or more initial dose(s) of the Fc fragment followed by one or more subsequent dose(s), wherein the mg/kg of subject body weight per day dose of the Fc fragment in the subsequent dose(s) exceeds the mg/kg of subject body weight per day dose of the Fc fragment in the initial dose(s). For example, the initial dose may be in the range from about 20 mg/kg of subject body weight per day to about 250 mg/kg of subject body weight per day (e.g., from about 50 mg/kg of subject body weight per day to about 200 mg/kg of subject body weight per day) and the subsequent dose may be in the range from about 250 mg/kg of subject body weight per day to about 1,000 mg/kg of subject body weight per day.

[085] In some embodiments, the Fc fragment of the present invention is an antagonist of CD16. The term "antagonist" is used in its normal sense in the art i.e., a chemical compound which prevents functional activation of a receptor (CD 16, in this case) by its agonist.

[086] The term "agonist" is known in the art as a chemical that binds to a receptor and activates the receptor to produce a biological response.

[087] In some embodiments, the Fc fragment of the present invention is a partial antagonist of a CD 16 Fc receptor. The term "partial antagonist" as used herein is an Fc fragment which is capable of specifically binding an Fc receptor wherein said binding elicits some effector functions but does not elicit other effector functions that are normally elicited by binding of an Fc of an IgG to the CD 16 Fc receptor.

[088] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. [089] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application. [090] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[091] In the description and claims of the present application, each of the verbs, "comprise," "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[092] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[093] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[094] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. EXAMPLES

[095] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document. Materials and Methods

Digestion of rituximab (IgGs) and enrichment of the Fc's fragment

[096] Rituximab Fc's fragments were prepared by papain digestion and enriched by protein G. Volume of 10 μΐ_^ rituximab (10 mg/ml; Roche) was treated with 15 μΐ_^ of papain (0.5 mg/ml; Sigma) in the presence of 75 μΐ_^ cysteine solution (5 mM; Sigma) for 1 hr at 37 °C.

The reaction was stopped by adding 100 μΐ_^ of iodoacetamide (5 mg/ml; Sigma). According to the manufacturer's instructions (GE healthcare, Germany) the Fc fragments were separated from the Fab fragments using protein G sepharose. Briefly, 1 volume of digested rituximab (100 μυ> diluted with 1 volume of binding buffer (20 mM sodium phosphate, pH 7.0) was applied onto a protein G column. After 1 h of incubation at room temperature under rotating conditions, the beads were washed, the Fc bounded fraction was eluted with 100 μΐ_^ of elution buffer (0.1 M glycine-HCl, pH 2.7), and the supernatants were collected into 1 M Tris-HCl, pH 8.5, to neutralize the Fc solutions to pH 7.5. Fc concentration was determined by Bradford assay (Bio-Rad, Hercules, CA). The Fc samples were immediately frozen for storage at -20 °C until thawed for injection or in vitro activity and inhibition assays.

Rituximab injection protocol

[097] Male and female mSODl^G93A mice and their age-matched littermates at 70 days of age were administered Fc rituximab or PBS as control via intrathecal injections. The BBB penetrability was increased 20 min prior to intrathecal injection; mice were weighed then Mannitol (20%: in the ratio of 1/20 (Mannitol volume ^L)/mouse weight (gr)); Baxter Healthcare Corporation) were injected intra-peritoneum (IP). Ten (10) min later mice were anesthetized using 100 μΐ_^ intra-muscular (IM) injection of Ketamin and Xylazine mixture (200 μL· of Ketamin with 100 μΐ_^ of Xylazine diluted up to 4 mL with PBS). After 10 min, when mice dazed, 100 μΐ_^ of diluted Fc-rituximab (5 μg/mL in PBS) were intrathecal injected into the cerebrospinal fluid ventricle, using 40 ⁰ folded 23G needles. After injection, mice were weighed and evaluated by neurological disability. Neurological score of four limbs was blindly performed by an independent physiotherapist using the scale of 0-5, with 0 being normal and 5 being completely paralyzed. Brain and spinal cord extraction

[098] Brains and spinal cords were extracted from a mice transcardially perfused with PBS. Tissues were fixed with 4% formaldehyde and cryoprotected in 30% sucrose solution. Finally, samples were frozen in OCT and cut into 18μιη sections for immunofluorescence.

In vivo imaging [099] In vivo imaging studies were conducted using the WIS® Lumina LT Series III preclinical in vivo imaging system. TNFa enzyme-linked immunosorbent assay (ELISA)

[0100] Brains and spinal cord were obtained from treated and untreated mice, and TNF-a was quantified with TNF-a ELISA kit (Biolegened) according to the manufacturer's instruction.

Gene expression [0101] Total RNA was isolated by the EZ-RNA kit (Biological Industries) according to the manufacturer's instruction, RNA was reverse transcribed into cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems) and cDNA was used for quantitative realtime PCR (qRT-PCR) analysis.

Flow cytometry General procedure

[0102] For fluorescence-activated cell sorting (FACS) analysis of single-cell samples (NSC-34, BW-CD16, BV2 or isolated glia cells), cells were harvested, washed and re- suspended in FACS buffer (2% FBS in PBS). The cells were plated on a 96-u-bottom plate at a concentration of 10⁵ cells per well (for NSC-34, BW-CD16 and BV2) or 6 x 10⁴ cells per well (glia). When intracellular markers were being detected, the cells were permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) following the manufacturer's instructions. After washing with FACS buffer, cells were incubated with primary antibodies for 1 h. Subsequently, cells were washed and stained with fluorophore-conjugated secondary antibody for 30 min in darkness. Finally, the cells were washed again and re-suspended in 200 μΐ FACS buffer for FACS analysis. All steps were performed on ice or in a chilled centrifuge (4 °C). The acquisition was performed using FACS Canto II (BD Biosciences), and fluorescence data was acquired using logarithmic amplification. Analysis was performed with FlowJo software (Three Star, Ashland, OR).

Antibodies [0103] Primary commercial antibodies used were as follows: rabbit anti-IBAl (1:600, WAKO), biotinylated anti-CD16 (ASH1975, 1: 100, Santa Cruz), rabbit anti-GFAP (1:500, Abeam), Alexa Fluor 488 anti-CDl lb (1:200, eBioscience), Alexa Fluor 647 anti-CDl lb (1:200, eBioscience), APC anti-CD 16/32 (1:200, eBioscience) and both Rituximab and Fc- Rituximab labeled with Alexa Flour™ 680 carboxylic acid (Invitrogen) and DyLight™ 488- NHS Ester (Thermo Fisher). Intact Rituximab and Fc-Rituximab were used at a concentration of 30 μg/ml. Secondary antibodies used were as follows: Alexa Fluor 647-conjugated Sterptavidin (1:1,800), APC-conjugated AffiniPure Goat anti-human IgG (1:200), Alexa Fluor 488-conjugated donkey anti-goat IgG (1:70) and Alexa Fluor 488-conjugated donkey anti- mouse IgG (1:200), all of Jackson Immuno-Research. Alexa Fluor 488-conjugated donkey anti- rabbit IgG ( 1 :250), Alexa Fluor 546-conjugated donkey anti-rabbit IgG ( 1 :250), both of Thermo Scientific. Phagocytosis

[0104] Purified primary microglial cells or BV2 were seeded onto coverslips at a density of 50,000 cells/cm². The cultures were grown in an incubator containing 5% C0₂ and 100% humidity at 37 °C. Microglia attached to the wells within 2 h of seeding. The wells were replenished with fresh, pre-warmed microglial culture medium DMEM/F12 after cells were attached to allow 24 h for the microglial cells recovering, after which the cells were ready for the phagocytosis assay the following day.

[0105] Fluorescent beads - aqueous green fluorescent latex micro-beads were pre- opsonized in FBS for 1 h at 37 °C. The ratio of beads to FBS was 1:5. The bead-containing FBS with DMEM were dilute to reach the final concentrations for beads and FBS in DMEM of 0.01% (v/v) and 0.05% (v/v), respectively.

[0106] Apoptotic NSC34 cells - apoptosis induction of cells was allowed using UV irradiation. NSC34 cells (1 x 10⁵) adhered to cell culture plate (24 well plate) were stained with CFSE and then exposed to UV irradiation for 15 min. Microglial conditioned culture media were replace with either beads- or NSC34 cells-containing DMEM and cultures were incubated at 37 °C for 1 h. For a well of a 24-well plate, the inventors added 250 μΐ beads or 1 x 10⁵ NSC34 apoptotic cells. Cultures were washed thoroughly with ice-cold PBS 5 times and then cells were fixed using 4% PFA for 15 min. the inventors visualized either green fluorescent beads or green apoptotic cells with the green channel and used the red channel for Ibal (microglial) staining. The inventors then counted microglia with 1, 2, 3, 4, 5, 6, 7...n beads within the cells by flow cytometry assessment.

BV-2 cell culture

[0107] The BV-2 murine microglial cell line was shown to exhibit phenotypic and functional properties comparable to those of primary microglia and are thus considered as a suitable model for in vitro studies of activated microglial cells. Briefly, BV-2 cells were grown and maintained in DMEM supplemented with 10% FCS a humidified atmosphere at 37 °C and 5% CO2 incubator. Hippocampal slice culture

[0108] Organotypic slice cultures are prepared according to the membrane interface method (Stoppini et al., (1991)). Brains are removed from sacrificed old (10-20 months) Alzheimer's disease (AD) mice after perfusion. Hippocampi of the AD mice are frozen then sagittal sections, 150 μιη thick, are dissected using vibratome instrument. Intact sections carefully selected under a dissection microscope are kept on slide glass in 6-well culture dish culture and supplemented with 1.2 ml medium of 0.5x MEM, 25% BME, 25% fetal bovine serum, 2 mM glutamax, 0.65% glucose; pH 7.2 in a humidified atmosphere at 37 °C and 5% C0₂. The medium is changed daily.

An ex-vivo uptake of β-amyloid aggregates by treated-BV2 cell line

[0109] Uptake experiments are performed according to the β-amyloid aggregates uptake method (Safaiyan et al., (2016)). BV2 cells, 1 x 10⁵ treated for 6 h with Fc of rituximab (1-20 μg/ml), intact IgGl of rituximab (1-20 μg/ml), lipopolysaccharide (LPS, 1 μg/ml) and untreated BV2 are subsequently added (2,000 cells/μΐ) to hippocampal slices (1 cm²) for additional 24 h in a humidified atmosphere at 37 °C and 5% CO2 in 0.5x MEM, 25% BME, 25% fetal bovine serum, 2 mM glutamax, 0.65% glucose; pH 7.2 medium. Several hippocampal slices without BV2 are used as control. A day later medium is washed out twice with PBS then changed by 4% paraformaldehyde (PFA) for fixation to accomplish immunohistochemistry.

Immunohistochemistry

[0110] For immunocytochemistry the fixed slices and BV2 cells are stained with primary antibodies of anti-P-amyloid aggregates (1:200), anti-Ibal (1: 1,000; Wako, Japan), anti-mouse APC-CDl lb (1:200; Biolegend). Incubation with secondary antibodies anti-rabbit Alexa fluore-488 (1:1,000) or anti-mouse Alexa fluore-647 (1: 1,000; Jackson Immunoresearch Ltd.) is followed by image acquisition using a Zeiss LSM 5 Exciter (Zeiss).

EXAMPLE 1

Effect of Fc-Rituximab on neurons

[0111] Neurons of spinal cord and brain of 136 days old mSODl^G93A mice treated with Fc-Rituximab or PBS, were double-stained for NeuroN (for labeling neurons) and nuclear DAPI (4',6-diamidino-2-phenylindole) and confocally imaged.

[0112] Representative confocal microscopic images of brain (Figs. 1A-1F) and SC (Figs.

1G-1L) showed a decrease in neurodegeneration (NeuN-) in both brain (Figs. 1A-1C) and SC

(Figs. 1G-1I) sections of mSODl^G93A mice treated with Fc-Rituximab in comparison to PBS. EXAMPLE 2

Distribution of Fc-Rituximab in mice's brains

[0113] The distribution of Fc-Rituximab in the brains of wild type and mSODl^G93A mice was examined. To this end, mice were subjected to in vivo fluorescence using IVIS imaging 2.5 hours after injection of labeled Fc-Rituximab with Alexa Fluor 680.

[0114] As seen in Fig. 2, brain from mSODl^^mice showed a higher signal intensity in comparison to WT mice brain.

EXAMPLE 3

Effect of Fc-Rituximab on microglia cytokine secretion [0115] The effect of Fc-Rituximab on TNF-a release in cultured primary glia cells was evaluated. To this end, mSODl^G93A and WT mice were treated with Fc-Rituximab or PBS and sacrificed at 120 days old (n=6), and the levels of TNF-a were measured using ELISA kit. Results demonstrated increased TNF-a release in microglia cells treated with FC-Rituximab (Figs. 3A-3B). [0116] Further, the effect of various treatment including Rituximab, Fc-Rituximab and LPS on TNF-a release in cultured primary microglia cells was compared. To this end, in day 95 (disease onset), primary glia cells were isolated from brains of sacrificed mSODl^G93A mice, then incubated over night with RPMI medium supplemented with inactivated fetal bovine serum (Blank), Fc-Rituximab (0.12 mg/ml), intact Rituximab (1 mg/ml) or LPS (n=3), and the levels of TNF-a were measured using ELISA kit.

[0117] The results demonstrated increased TNF-a release in cultured primary microglia cells using Fc-Rituximab (Fig. 4).

EXAMPLE 4

Gene expression profile in microglia cells after Fc-Rituximab injection [0118] In order to evaluate the expression of inflammatory genes, glia cells were isolated from both mSODl and WT mice 7 days after injection of Fc-rituximab or PBS. Inflammatory gene expression levels were analyzed by using qPCR, and TAQ-MAN primers. All samples were normalized to GAPDH housekeeping gene and to mSODl mice injected with PBS.

[0119] The results demonstrated an increase in IL-1 beta and CCL2 gene expression in brain (Figs. 5B and 5D) and an increase in IL-1 beta and CCL2 in spinal cord (Figs. 5 A and 5C). Further support for the activation of cells in either the brain, spinal cord or microglia as demonstrated by the expression of relevant cytokines are given in figs. 9-10 and 12.

EXAMPLE 5

The Fc- Rituximab improves clearing debris activity of microglial cells [0120] In order to evaluate the effect of Fc-Rituximab on the phagocytic activity of microglial cells, phagocytosis of apoptotic NSC34 by cells of a microglial cell line (BV-2 cells) was examined. To this end, BV-2 cells were incubated for 16 hours in RPMI medium supplemented with inactivated fetal bovine serum (Blank), Fc-Rituximab (0.116 mg/ml) or different dilutions of intact Rituximab (0.25-1 mg/ml). Next, apoptotic and stained motor neuron line of NSC34 cells were added to the BV-2 cells for additional 3 h. Intracellular staining was performed with CFSE and irradiation by UV until cells turned into apoptotic cells. Apoptosis was measured by 7AAD nuclei staining. Fluorescence microscope images were obtained (Figs. 6A-6C) and the phagocytic index was calculated by measuring the number of BV-2 cells with CFSE debris to total number of BV-2 cells per field (Fig. 6D). EXAMPLE 6

Dispersity and clearance of the injected Fc-Rituximab

[0121] The inventors investigated the dispersity and clearance of the Fc in the CNS with time. Briefly, mice were intrathecally injected to the CSF once with Fc-Rituximab-labeled with Alexa Flour™ 680. The fluorescent images post injection demonstrated that the Fc labeled to AF-680 scatters in the CNS 2 h and 4 h post injection (Fig. 7A) but was not detected 8 h, and 10 h (data not shown) after injection in both spinal cords (SCs) and brains. The data also indicated that the unbound Fc was cleared from the CNS over the period of 8 h. Next the inventors assessed the distribution of the bound Fc in the CNS tissues. Results showed distribution of the Fc in the brain stem indicating that the Fc binds to certain cells in the CNS. Specifically, co-localization of Fc with microglia cells was detected in the brain tissue sections, showing that the Fc bound directly to microglia cells in the CNS (Fig. 7B). Fc binding was further confirmed in vitro by flow cytometry analysis where Fc-bound cells were doubled stained with secondary antibodies against human Fc and anti-CD l ib or -Ibal antibodies (Fig. 7C-D). EXAMPLE 7

Effects of single Fc-Rituximab injection on weight, neurological score and life

expectancy

[0122] Next, the inventors examined whether the Fc-Rituximab prolongs life expectancy. Since weight loss also correlates with the progression of the disease, mice were weighed and evaluated for neurological disability. Male mice injected with Fc-Rituximab were positively affected. Remarkably, Fc-Rituximab significantly increased male lifespan by an average of 21 days (Fig. 8B). Furthermore, there was a delay in both disease onset and progression. Results showed a significant difference in weight loss between treated and untreated mice (Fig. 8B). The female group revealed a less dramatic effect, although treated individuals were characterized by a reduced weight loss and improved neurological score, compared with the PBS-injected control (Fig. 8D). The female lifespan increased only by an average of 6 day. The symptoms initiated and progressed at the same incidence as the control, and the difference in weight lost was seen only towards the end of the experiment. EXAMPLE 8

[0123] To test the ability of microglia to remove pathogens as well as clearance of toxic molecules from the CNS the inventors investigated the ability of the microglia to respond to an external stimulation through Toll-like receptor 4 (TLR4) signaling in vitro. Consequently, glia cells were isolated from SC and brain tissues taken from 120-days old mSODl mice injected with Fc-Rituximab or PBS, and WT treated with Fc-Rituximab. Microglia stimulation was achieved by incubating the cells with lipopolysaccharides (LPS). Levels of TNFa were measured by ELISA (Fig. 11A-B). Data revealed a significant difference in the levels of secreted TNFa between the Fc and the control group in mSODl mice. Interestingly, no significant difference was measured between WT and mSODl mice treated with Fc. Glia cells were further isolated from SC tissues taken from 95-days old untreated mSODl male mice. Levels of TNFa were measured in the supernatants of the isolated glial cells after an overnight incubation with Fc, intact Rituximab or without treatment. Data revealed a significant difference in the levels of secreted TNFa between the Fc and the Rituximab and control groups. [0124] The ability of B V2 or primary microglia cells to phagocytose was further measured in vitro following treatment with Fc, intact Rituximab or without treatment (Fig. 11C-F).

Phagocytosis was performed by counting cells uptake fluorescent micro-beads or UV-irradiated apoptotic NSC34 cells. Results show a significant difference in bead and apoptotic NSC34 cell uptake by BV2 cells treated with Fc relative to intact Rituximab and control groups. High rate of uptake beads by BV2 was further documented after Fc treatment compared to the other groups. Bead uptake trend was consistent in primary microglia isolated from WT mice as in BV2. Counting cells with beads further displayed high number of cells treated by Fc compared to untreated cells (control).

EXAMPLE 9

Ex vivo phagocytosis of β-amyloid aggregates accumulated in hippocampus of

Alzheimer's disease mice by immortalized BV2 microglia

[0125] In order to evaluate microglia cells ability to phagocytize β-amyloid aggregates, BV2 cells (1 x 10⁵) treated for 6 h with Fc of rituximab (1-20 μg/ml), intact IgGl of rituximab (1-20 μg/ml), lipopolysaccharide (LPS, 1 μg/ml) or left untreated are subsequently added (2,000 cells/μΐ) to hippocampal slices of the AD mice (1 cm²) for 24 h (37 °C and 5% C0₂). Hippocampal slices without BV2 are used as control. A day later medium is washed out and preparates are then fixated. The fixed slices and BV2 cells are initially incubated with primary antibodies and subsequently stained with secondary antibodies. Thereafter, image acquisition is followed.

[0126] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

1. A method for enhancing phagocytic activity in a subject in need thereof, the method comprises administering to said subject a therapeutically effective amount of an immunoglobulin Fc fragment, wherein said Fc fragment is characterized by an increased binding affinity to an Fc receptor, thereby enhancing phagocytic activity of the microglia cells in said subject.

2. A method for treating a neuronal disorder in a subject in need thereof, the method comprises administering to said subject a therapeutically effective amount of an immunoglobulin Fc fragment, wherein said Fc fragment is characterized by increased binding affinity to an Fc receptor on microglia cells, thereby treating the neuronal disorder in said subject.

3. The method of any one of claims 1 or 2, wherein said Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1 or derivative, a fragment or an analog thereof.

4. The method of any one of claims 1 or 2, wherein said Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof.

5. The method of any one of claims 1 or 2, wherein said Fc fragment comprises two polypeptides, each polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1.

6. The method of any one of claims 1 or 2, wherein is said Fc fragment is an antagonist of an Fc receptor.

7. The method of any one of the preceding claims, wherein said Fc fragment comprises a glycosylated asparagine amino acid residue.

8. The method of claim 7, wherein said glycosylated asparagine is located at amino acid position 297 of the IgG heavy chain (N297).

9. The method of any one of claims 7 or 8, wherein said N297 comprises a bisecting N-acetyl glucosamine (GlcNAc).

10. The method of claim 1, wherein said phagocytic activity is phagocytic activity of microglia.

11. The method of claim 1, wherein said Fc receptor is an Fc receptor expressed on microglia cells.

12. The method of any one of claims 1-11, wherein said Fc receptor is CD16.

13. The method of any one of claims 1 or 2, wherein said increased binding affinity is characterized by a dissociation constant (Kd) between 0.1-10 micro molar (μΜ).

14. The method of claim 2, wherein said neuronal disorder is a neurodegenerative disease.

15. The method of claim 12, wherein said neurodegenerative disease is selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and age-related macular degeneration (AMD).

16. A pharmaceutical composition comprising an immunoglobulin Fc fragment and a pharmaceutically acceptable carrier, wherein said Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof, for use in enhancing microglia phagocytosis.

17. A pharmaceutical composition comprising an immunoglobulin Fc fragment and a pharmaceutically acceptable carrier, wherein said Fc fragment comprises a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2 or derivative, a fragment or an analog thereof, for use in treating a neurodegenerative disease selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and age-related macular degeneration (AMD).