WO2018154580A1 - Polypeptides à base de fc et leur utilisation - Google Patents

Polypeptides à base de fc et leur utilisation Download PDF

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Publication number
WO2018154580A1
WO2018154580A1 PCT/IL2018/050209 IL2018050209W WO2018154580A1 WO 2018154580 A1 WO2018154580 A1 WO 2018154580A1 IL 2018050209 W IL2018050209 W IL 2018050209W WO 2018154580 A1 WO2018154580 A1 WO 2018154580A1
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Prior art keywords
fragment
cells
disease
amino acid
microglia
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PCT/IL2018/050209
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English (en)
Inventor
Rachel Glicklis LICHTENSTEIN
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B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
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Publication of WO2018154580A1 publication Critical patent/WO2018154580A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to immunoglobulin derived Fc fragments and their use in treatment of neurological diseases.
  • 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.
  • the present invention relates to methods and compositions for treating neurological diseases in a subject in need thereof.
  • the present invention provides a method for enhancing phagocytic activity of microglia.
  • a method for enhancing phagocytic activity in a subject in need thereof 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.
  • a method for treating a neuronal disorder in a subject in need thereof 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.
  • 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.
  • the glycosylated asparagine is located at amino acid position 297 of the IgG heavy chain (N297).
  • N297 comprises a bisecting N-acetyl glucosamine (GlcNAc).
  • the phagocytic activity is phagocytic activity of microglia.
  • the Fc receptor is an Fc receptor expressed on microglia cells.
  • the Fc receptor is CD 16.
  • increased binding affinity is characterized by a dissociation constant (Kd) between 0.1-10 micro molar ( ⁇ ).
  • the neuronal disorder is a neurodegenerative disease.
  • 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).
  • 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.
  • 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).
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • HD Huntington's disease
  • AMD age-related macular degeneration
  • 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 ⁇ .
  • 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 G93A mice showed a higher signal intensity in comparison to WT mice brains in both male and female.
  • 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).
  • 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.
  • 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 SODl 09 ⁇ 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 SODl 09 ⁇ males 50 days after Fc and PBS administration (D). Data are mean + s.e.m.
  • 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.
  • 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.
  • 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.
  • Figs. 11A-11F describe TNFa secretion and phagocytosis by isolated or immortalized microglia.
  • 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- ⁇ , 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.
  • the present invention relates to methods and compositions for treating neurological diseases in a subject in need thereof.
  • the present invention provides a method for enhancing phagocytic activity of microglia.
  • the method comprises administering to a subject in need thereof an isolated antibody Fc fragment, as described herein.
  • said Fc fragment is an antagonist of CD 16.
  • the CD 16 Fc receptor is selected from: FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b).
  • the Fc fragments of rituximab increases microglia phagocytosis.
  • 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).
  • TNF alpha tumor necrosis factor alpha
  • C-C motif chemokine ligand 2
  • 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.
  • 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.
  • 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.
  • the immunoglobulin Fc fragment of the invention are devoid of a Fab region.
  • 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.
  • 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.
  • the hinge region of the Fc fragment of the present invention has a length of at least 12 amino acids.
  • the hinge region of the Fc fragment of the present invention has a length of at least 15 amino acids.
  • the hinge region of the Fc fragment of the present invention has a length of between 12-62 amino acids.
  • the hinge region of the Fc fragment of the present invention has a length of between 15-62 amino acids.
  • 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.
  • a human-derived Fc fragment is a recombinant immunoglobulin Fc fragment that is obtained from a microorganism. Fc glycoform modification
  • 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.
  • carbohydrate moieties such as glycosylation with oligosaccharides.
  • glycosylation 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.
  • 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.
  • glycoform modification of the Fc fragment of the present invention increases an effector function.
  • glycoform modification of the Fc fragment of the present invention increases binding affinity to CD16.
  • 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.
  • 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.
  • 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.
  • residue 297 refers to the asparagine at location 301 of SEQ ID NO: 2.
  • residue 297 refers to the asparagine at location 130 of SEQ ID NO: 1.
  • said glycosylation at amino acid residue 297 is of a bisecting N-acetyl glucosamine (GlcNAc).
  • said bisecting-GlcNAc lacks a core fucose.
  • said glycosylation is A2BG2.
  • the altered glycosylation comprises an increased level of bisected complex residues in the Fc fragment.
  • the altered glycosylation comprises a reduced level of fucose residues in the Fc fragment.
  • microglia refers to the smallest of the glial cells that can act as phagocytic cells, cleaning up CNS debris.
  • the methods and compositions of the present invention increase the secretion of pro-inflammatory cytokines from microglia.
  • pro-inflammatory cytokines include TNF alpha and CCL2.
  • the CD 16 binding Fc fragment of the present invention is an Fc variant having affinity to CD 16.
  • Fc variant 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.
  • 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.
  • 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.
  • 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.
  • 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 ( ⁇ ).
  • 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.
  • 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.
  • the Fc variant increases ADCC by at least 10% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases ADCC by between 10% -50% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases ADCC by between 50%- 100% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases ADCC by between 100%-500% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases ADCC by more than 500% compared to WT Fc fragment as measured by EC50 values.
  • 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.
  • the Fc variant increases CDC by at least 10% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases CDC by between 10%-50% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases CDC by between 50%- 100% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases CDC by between 100% -500% compared to WT Fc fragment as measured by EC50 values.
  • the Fc variant increases CDC by more than 500% compared to WT Fc fragment as measured by EC50 values.
  • 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.
  • 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.
  • 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.
  • 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.
  • the optimized property of the Fc variant of the present invention is increased or reduced affinity for any Fc receptor.
  • 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.
  • the Fc variants are optimized to possess reduced affinity for the human inhibitory receptor FcyRIIb.
  • the alteration of affinity increases an effector function.
  • 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.
  • 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.
  • 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.
  • the Fc variant of the present invention may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • the Fc fragment of the present invention is an Fc fusion.
  • 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.
  • targets may be any molecule, preferably an extracellular receptor that is implicated in disease.
  • 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.
  • 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.
  • 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.
  • 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 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 1).
  • 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:
  • the CD 16 binding Fc fragment of the present invention is obtained by papain cleavage of an engineered humanized anti CD20 immunoglobulin.
  • the Fc fragment of the present invention is a synthetic peptide generated by methods known in the art.
  • the peptides of the present invention can be purified so that the peptides will be substantially pure when administered to a subject.
  • substantially pure refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it.
  • 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.
  • 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.
  • conjugates comprising peptides of the invention and a different protein can be made by protein synthesis.
  • conjugates comprising peptides of the invention and a different protein can be made by use of a peptide synthesizer.
  • 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.
  • 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.
  • the linkers are comprised of at least one amino acid residue.
  • the linkers can be of 40 or more residues.
  • the linkers are comprised of 1 to 10 residues.
  • amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
  • derivative 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.
  • 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.
  • analog 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.
  • 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.
  • 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.
  • 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.
  • wild-type (WT) Fc fragment 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.
  • 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.
  • the neuronal disorder is a neurodegenerative disorder.
  • 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).
  • the neuronal disorder is a neurodegenerative disorder other than amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • 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.
  • administering 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.
  • a therapeutically effective dose of the Fc fragment of the present invention is administered.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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
  • 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.
  • administration of the pharmaceutical composition comprising the Fc fragment of the present invention 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.
  • intravenously intravenously, intranasally, intraotically, transdermally, topically
  • topically e.g., gels, salves, lotions, creams, etc.
  • intraperitoneally e.g., intramuscularly, intrapulmonary (e.g., AERx® inhalable technology commercially available from Aradigm, or Inhance® pulmonary
  • 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).
  • 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.
  • the Fc fragment of the present invention is an antagonist of CD16.
  • 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.
  • agonist is known in the art as a chemical that binds to a receptor and activates the receptor to produce a biological response.
  • the Fc fragment of the present invention is a partial antagonist of a CD 16 Fc receptor.
  • 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.
  • 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.
  • Rituximab Fc's fragments were prepared by papain digestion and enriched by protein G. Volume of 10 ⁇ ⁇ rituximab (10 mg/ml; Roche) was treated with 15 ⁇ ⁇ of papain (0.5 mg/ml; Sigma) in the presence of 75 ⁇ ⁇ cysteine solution (5 mM; Sigma) for 1 hr at 37 °C.
  • the reaction was stopped by adding 100 ⁇ ⁇ 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 ⁇ > diluted with 1 volume of binding buffer (20 mM sodium phosphate, pH 7.0) was applied onto a protein G column.
  • the beads were washed, the Fc bounded fraction was eluted with 100 ⁇ ⁇ 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.
  • 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).
  • IP intra-peritoneum
  • mice Ten (10) min later mice were anesthetized using 100 ⁇ ⁇ intra-muscular (IM) injection of Ketamin and Xylazine mixture (200 ⁇ L ⁇ of Ketamin with 100 ⁇ ⁇ of Xylazine diluted up to 4 mL with PBS). After 10 min, when mice dazed, 100 ⁇ ⁇ of diluted Fc-rituximab (5 ⁇ g/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
  • 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 18 ⁇ sections for immunofluorescence.
  • 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.
  • FACS fluorescence-activated cell sorting
  • 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 FlourTM 680 carboxylic acid (Invitrogen) and DyLightTM 488- NHS Ester (Thermo Fisher).
  • Intact Rituximab and Fc-Rituximab were used at a concentration of 30 ⁇ g/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.
  • 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.
  • NSC34 cells - apoptosis induction of cells was allowed using UV irradiation.
  • NSC34 cells (1 x 10 5 ) 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.
  • the inventors added 250 ⁇ beads or 1 x 10 5 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 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
  • 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 ⁇ 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% C0 2 . The medium is changed daily.
  • Uptake experiments are performed according to the ⁇ -amyloid aggregates uptake method (Safaiyan et al., (2016)).
  • BV2 cells 1 x 10 5 treated for 6 h with Fc of rituximab (1-20 ⁇ g/ml), intact IgGl of rituximab (1-20 ⁇ g/ml), lipopolysaccharide (LPS, 1 ⁇ g/ml) and untreated BV2 are subsequently added (2,000 cells/ ⁇ ) to hippocampal slices (1 cm 2 ) 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.
  • 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).
  • mice were subjected to in vivo fluorescence using IVIS imaging 2.5 hours after injection of labeled Fc-Rituximab with Alexa Fluor 680.
  • 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.
  • the Fc- Rituximab improves clearing debris activity of microglial cells [0120]
  • phagocytosis of apoptotic NSC34 by cells of a microglial cell line was examined.
  • 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).
  • 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
  • mice were intrathecally injected to the CSF once with Fc-Rituximab-labeled with Alexa FlourTM 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.
  • SCs spinal cords
  • the data also indicated that the unbound Fc was cleared from the CNS over the period of 8 h.
  • 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
  • TLR4 Toll-like receptor 4
  • 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).
  • BV2 cells (1 x 10 5 ) treated for 6 h with Fc of rituximab (1-20 ⁇ g/ml), intact IgGl of rituximab (1-20 ⁇ g/ml), lipopolysaccharide (LPS, 1 ⁇ g/ml) or left untreated are subsequently added (2,000 cells/ ⁇ ) to hippocampal slices of the AD mice (1 cm 2 ) for 24 h (37 °C and 5% C0 2 ). 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.

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Abstract

La présente invention concerne des méthodes comprenant l'administration de fragments Fc dérivés de l'immunoglobuline à un sujet atteint d'une maladie neurodégénérative. L'invention concerne en outre des compositions et des méthodes pour améliorer la phagocytose des microglies à l'aide desdits fragments Fc dérivés de l'immunoglobuline.
PCT/IL2018/050209 2017-02-24 2018-02-22 Polypeptides à base de fc et leur utilisation WO2018154580A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006116369A2 (fr) * 2005-04-22 2006-11-02 Genentech, Inc. Méthode pour traiter la démence ou la maladie d'alzheimer
WO2013084236A1 (fr) * 2011-12-05 2013-06-13 Ben-Gurion University Of The Negev Research And Development Authority Diagnostic et méthodes thérapeutiques et leur application dans la sclérose latérale amyotrophique (als)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006116369A2 (fr) * 2005-04-22 2006-11-02 Genentech, Inc. Méthode pour traiter la démence ou la maladie d'alzheimer
WO2013084236A1 (fr) * 2011-12-05 2013-06-13 Ben-Gurion University Of The Negev Research And Development Authority Diagnostic et méthodes thérapeutiques et leur application dans la sclérose latérale amyotrophique (als)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANTHONY, ROBERT M. ET AL.: "Novel roles for the IgG Fc glycan", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 1253, 2012, pages 170 - 180, XP055536615, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.2011.06305.x/full> [retrieved on 20170614] *
OKUN, EITAN ET AL.: "Involvement of Fc receptors in disorders of the central nervous system", NEUROMOLECULAR MEDICINE, vol. 12, no. 2, 2010, pages 164 - 178, XP055529671, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2878892> [retrieved on 20180501] *

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