WO2015057939A1 - Anticorps anti-s1p4 et leurs utilisations - Google Patents

Anticorps anti-s1p4 et leurs utilisations Download PDF

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Publication number
WO2015057939A1
WO2015057939A1 PCT/US2014/060858 US2014060858W WO2015057939A1 WO 2015057939 A1 WO2015057939 A1 WO 2015057939A1 US 2014060858 W US2014060858 W US 2014060858W WO 2015057939 A1 WO2015057939 A1 WO 2015057939A1
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Prior art keywords
antibody
antigen
amino acid
binding fragment
seq
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PCT/US2014/060858
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English (en)
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Sha Mi
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Biogen Idec Ma Inc.
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Publication of WO2015057939A1 publication Critical patent/WO2015057939A1/fr

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • This invention relates generally to antibodies that bind to the sphingosine-1 -phosphate receptor 4 (S 1P4) and uses thereof.
  • Neurodegenerative diseases are defined as hereditary and sporadic conditions that are characterized by progressive nervous system dysfunction. These disorders are often associated with atrophy of the affected central or peripheral structures of the nervous system. They include diseases such as Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Huntington's Disease, Prion Diseases, demyelinating diseases such as Multiple Sclerosis, and motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease).
  • diseases such as Alzheimer's Disease and other dementias, Brain Cancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, Genetic Brain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke, Parkinson's Disease, Huntington's Disease, Prion Diseases, demyelinating diseases such as Multiple Sclerosis, and motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS or Lou
  • Demyelinating diseases are disorders of the central or peripheral nervous system that involve destruction of myelin.
  • Myelin provides discontinuous insulation along axons that leads to a reduction in the threshold for neuronal activation and an increase in axonal conduction velocity (Nat. Neurosci. 10, 1351-1354 (2007)).
  • the central nervous system is replete with oligodendrocyte progenitor cells (OPCs) that can differentiate into mature, myelinating oligodendrocytes (Nat. Rev. Neurosci. 9, 839-855 (2008)).
  • OPCs oligodendrocyte progenitor cells
  • oligodendrocytes Development of oligodendrocytes is characterized by three major stages: (1) A2B5 + progenitor cells, (2) immature platelet-derived growth factor receptor a (PDGFRa) + and 04 + oligodendrocytes, and (3) mature myelinating oligodendrocytes that express myelin-associated glycoprotein (MAG), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG).
  • MAG myelin-associated glycoprotein
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • oligodendrocyte lineage cells The maturation of oligodendrocyte lineage cells is highly regulated during development and tightly linked to survival signals. However, the complete maturation of these cells is frequently blocked in inflammatory demyelinated lesions, such that the remyelination process is curtailed (Chang et al, N. Engl. J. Med. 346, 165-173 (2002)).
  • the most common demyelinating disease is multiple sclerosis. It is the most frequently occurring non-traumatic neurological disease affecting young adults in the Western world (Compston and Coles, Lancet 359(9313): 1221-31 (2002); Hauser and Oksenberg, Neuron, 52(1): 61-76 (2006)).
  • the disease has a complex and varied pathology but is typically characterized by acute multi-focal CNS autoimmune-mediated demyelination,
  • S1P4 is an important regulator of oligodendrocyte differentiation and myelination. Specifically, blocking S 1P4 activity promotes oligodendrocyte differentiation and myelination.
  • the present disclosure provides antibodies or antigen binding fragments thereof that specifically bind S1P4 and promote oligodendrocyte differentiation and myelination. Such antibodies and antigen binding fragments thereof are useful in treating or preventing neurological diseases. For example, such antibodies and antigen binding fragments thereof are useful in treating or preventing demyelinating diseases such as multiple sclerosis (MS) and motor neuron diseases such as amyotrophic lateral sclerosis (ALS).
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • the disclosure features an isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4, when expressed on the surface of a cell, at an epitope within amino acid residues 23-50 of SEQ ID NO: 1.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4, when expressed on the surface of a cell, and competes with the 1F1 1 antibody for binding to human S1P4.
  • the antibody or antigen-binding fragment thereof binds non-dentatured S1P4. In another embodiment, the antibody or antigen-binding fragment thereof binds both non-dentatured and denatured S 1P4.
  • the disclosure features an isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4, wherein the antibody or the antigen- binding fragment thereof comprises a heavy chain complementarity determining region 1 (CDRl), a heavy chain CDR2, and a heavy chain CDR3.
  • the heavy chain CDRl comprises the amino acid sequence set forth in SEQ ID NO: 1 1 or the amino acid sequence set forth in SEQ ID NO: 1 1 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions.
  • the heavy chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO : 13 or the amino acid sequence set forth in SEQ ID NO : 13 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions.
  • the heavy chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 15 or the amino acid sequence set forth in SEQ ID NO: 15 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions.
  • the antibody or antigen-binding fragment thereof contains the heavy chain CDRl comprising the amino acid sequence set forth in SEQ ID NO: 1 1; the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13; and the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15.
  • the antibody or antigen-binding fragment thereof comprises a light chain CDRl, a light chain CDR2, and a light chain CDR3.
  • the light chain CDRl comprises the amino acid sequence set forth in SEQ ID NO: 17 or the amino acid sequence set forth in SEQ ID NO: 17 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions;
  • the light chain CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions;
  • the light chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO:21 or the amino acid sequence set forth in SEQ ID NO:21 with a substitution at three or fewer, two or fewer, three, two, or one amino acid positions.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 1; the heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13; the heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; the light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17; the light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19; and the light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4 and comprises a heavy chain variable domain (VH) that is at least 80% identical to the amino acid sequence of the VH domain of 1F1 1 (SEQ ID NO:7).
  • VH heavy chain variable domain
  • the VH domain is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VH domain of 1F11 (SEQ ID NO:7).
  • the VH domain is identical to the amino acid sequence of the VH domain of 1F1 1.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:39.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) that is at least 80% identical to the amino acid sequence of the VL domain of IF 1 1 (SEQ ID NO:9).
  • the VL domain is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL domain of IF 11 (SEQ ID NO:9).
  • the VL domain is identical to the amino acid sequence of the VL domain of 1F1 1.
  • the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO:41.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4 and comprises a heavy chain variable domain (VH) that is at least 90% identical to the amino acid sequence of the VH domain of 1F1 1 (SEQ ID NO: 7) and a light chain variable domain (VL) that is at least 90% identical to the amino acid sequence of the VL domain of 1F1 1 (SEQ ID NO:9).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the heavy chain variable domain (VH) is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VH domain of 1F11 (SEQ ID NO:7).
  • the light chain variable domain (VL) is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL domain of 1F11 (SEQ ID NO:9).
  • the isolated antibody or antigen-binding fragment thereof that selectively binds to human S1P4 comprises a heavy chain variable domain (VH) that is at least 95% identical to the amino acid sequence of the VH domain of IF 11 (SEQ ID NO: 7) and a light chain variable domain (VL) that is at least 95% identical to the amino acid sequence of the VL domain of 1F1 1 (SEQ ID NO:9).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the heavy chain comprises the amino acid sequence of SEQ ID NO:39
  • the light chain comprises the amino acid sequence of SEQ ID NO:41.
  • the isolated antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. In other embodiments, the isolated antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain constant region. In certain embodiments, the light chain constant region is a human constant region. In some embodiments, the isolated antibody or antigen- binding fragment thereof comprises a heavy chain constant region. In certain embodiments, the heavy chain constant region is a human constant region.
  • the antibody has a heavy chain isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, IgM. In certain embodiments, the antibody has a light chain isotype that is ⁇ or ⁇ . In some embodiments, the antigen-binding fragment is selected from the group consisting of an Fab, an Fab', an F(ab')2, an Fv, a diabody, an scFv, an sc(Fv)2, a domain antibody (dAb), and a nanobody.
  • the antibody or antigen- binding fragment thereof is conjugated to a toxin, a radionuclide, a fluorescent label, polyethylene glycol, a cytotoxic agent, a miRNA, a miRNA mimic, an anti-miR, a ribozyme, a siRNA, or a small molecule drug.
  • the disclosure features a pharmaceutical composition comprising any one or more of the antibodies or antigen-binding fragments thereof described herein.
  • the disclosure provides a monoclonal antibody preparation comprising, as the only molecular species of antibody or antigen-binding fragment thereof in the preparation, an antibody or antigen-binding fragment thereof described herein.
  • the disclosure features an isolated cell that produces an antibody or antigen-binding fragment thereof described herein.
  • the disclosure provides a method of detecting a S1P4 protein in a test sample.
  • the method involves contacting a test sample with an antibody or antigen- binding fragment thereof described herein; and detecting the presence of an immune complex between the antibody or antigen-binding fragment thereof and a protein in the test sample.
  • the disclosure features a method of making an antibody or an antigen-binding fragment thereof.
  • the method involves expressing the desired antibody or the antigen-binding fragment thereof described herein in a host cell; and isolating the antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is a IF 1 1 antibody or an antigen-binding fragment thereof.
  • the antibody is a IF 1 1 IgG antibody.
  • antibody is a IF 1 1 IgG2 antibody.
  • the disclosure features a method of treating a demyelinating disorder in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the demyelinating disorder is a central nervous system (CNS) demyelinating disorder.
  • CNS central nervous system
  • the CNS demyelinating disorder is selected from the group consisting of multiple sclerosis, Tabes Dorsalis, transverse myelitis, neuromyelitis optica (NMO), the CIS syndrome of CNS demyelination, progressive multifocal leukoencephalophathy, optic neuritis, and a leukodystrophy.
  • the disclosure features a method of treating multiple sclerosis in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the multiple sclerosis is relapsing remitting multiple sclerosis.
  • the multiple sclerosis is primary progressive multiple sclerosis.
  • the multiple sclerosis is secondary progressive multiple sclerosis.
  • the multiple sclerosis is progressive relapsing multiple sclerosis.
  • the disclosure features a method of treating amyotrophic lateral sclerosis in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the disclosure provides a method of promoting myelination or remyelination in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the disclosure features a method of promoting oligodendrocyte progenitor cell (OPC) differentiation in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • OPC oligodendrocyte progenitor cell
  • the disclosure features a method of promoting motor neuron survival in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the disclosure provides a method of blocking axon degeneration in a human subject in need thereof.
  • the method comprises administering to the human subject in need thereof an effective amount of an antibody or the antigen-binding fragment thereof, a pharmaceutical composition, or a monoclonal antibody preparation described herein.
  • the antibody or antigen- binding fragment thereof is a IF 1 1 antibody or an antigen-binding fragment thereof.
  • the antibody is a IF 1 1 IgG antibody.
  • antibody is a IF 1 1 IgG2 antibody.
  • the disclosure provides a method for screening for S1P4 antagonists. Such antagonists are useful in treating or preventing demyelinating diseases.
  • the method comprises contacting an oligodendrocyte precursor cell or cell line that expresses S1P4 with a test agent and determining whether the test agent increases the expression level of myelin basic protein, myelin associated glycoprotein, and/or myelin oligodendrocyte glycoprotein.
  • a test agent that increases the expression level of myelin basic protein, myelin associated glycoprotein, and/or myelin oligodendrocyte glycoprotein compared with a negative control is determined to be an S 1P4 antagonist, whereas a test agent that does not increase expression level of one or more these proteins compared with the negative control is determined to not be an S1P4 antagonist.
  • a test agent that increases the expression level of myelin basic protein, myelin associated glycoprotein, and/or myelin oligodendrocyte glycoprotein to the same extent or to 40%-99% of the level compared with a positive control e.g., an antibody that is known to increase the expression level of one or more of these proteins; e.g., IF 11
  • a positive control e.g., an antibody that is known to increase the expression level of one or more of these proteins; e.g., IF 11
  • the test agent is an antibody or antigen-binding fragment thereof.
  • the test agent is an siRNA.
  • the test agent is a small molecule drug.
  • Figure 1 is a bar graph depicting S1P4 mRNA expression in neuronal cells.
  • the graph shows the results of quantitative PCR analysis of first-strand cDNA synthesized from RNA obtained from rat neuronal cells. Rat actin mRNA was amplified as an internal control. The abundance of S 1P4 mRNA is presented relative to the normalized amount in A2B5 + OPCs (the mRNA level of which was set as 1).
  • Figure 2A is a bar graph showing expression of S 1P4 mRNA in rat neuronal cells.
  • the graph depicts the results of quantitative PCR analysis of first-strand cDNA synthesized from RNA obtained from rat Schwann cells, DRG neurons, OPC, and motor neurons.
  • Rat actin mRNA was used as an internal control.
  • the abundance of S1P4 mRNA is presented relative to the normalized amount in DRG (the mRNA level of which was set as 1).
  • Figure 2B provides a series of immunocytochemistry images of cultured motor neurons stained with anti-SlP4 and anti-microtubule-associated protein 2 (MAP2) antibodies. S1P4 and MAP2 co-localize in the motor neurons.
  • MAP2 anti-microtubule-associated protein 2
  • Figure 3 comprises two bar graphs depicting S1P4 expression in human neuronal cells.
  • the graphs shows the results of quantitative PCR analysis of first-strand cDNA synthesized from RNA obtained from primary neuron, oligodendrocyte (OPC), and other human cell lines.
  • Human actin mRNA was amplified as an internal control.
  • the abundance of S1P4 mRNA is presented relative to the normalized amount in SK-N-MC (the mRNA level of which was set as 1).
  • the abundance of mRNA is presented relative to the normalized amount of SIP 1.
  • Figure 4 is a bar graph showing S1P4 expression in human tissues.
  • the graph depicts quantitative PCR analysis of first-strand cDNA synthesized from RNA obtained from human tissues (Clontech). Human actin mRNA was amplified as an internal control. The abundance of S1P4 mRNA is presented relative to the normalized amount in the prostate (the mRNA level of which was set as 1).
  • Figure 5A is an image of a Western blot performed on extracts from
  • Figure 5C is a bar graph that shows the effect of S1P4 RNAi on the mRNA levels of different SIP receptors.
  • S1P4 siRNA shows specificity in knocking down S1P4 mRNA and not the other four S IP receptors.
  • Figure 7 is an image of a Western blot performed on extracts from oligodendrocytes that were transfected with individual plasmid carrying either full length (FL) or dominant negative (DN) versions of the indicated genes (i.e., S1P1, S1P4, S1P5, and LINGO-1). All the plasmid constructs contain the GFP gene. Western blotting with anti-MBP and anti-MAG antibodies was used to detect oligodendrocyte differentiation, while GFP (plasmid control) and ⁇ -actin (cell number control) were the internal controls after the transfection. LINGO-1 DN was used as a positive control for OPC differentiation.
  • FL full length
  • DN dominant negative
  • Figure 8B is an image of a Western blot of extracts from wildtype and S1P4 knock out (KO) oligodendrocytes using anti-MAG, anti-MBP, and anti- -actin (internal control) antibodies.
  • Figure 10 is an image of a Western blot performed on extracts of oligodendrocytes infected with lentivirus containing S 1P4 FL, S1P4 DN, or control GFP that were co-cultured with DRG neurons also infected with individual viruses as indicated. MBP and MOG Western blotting was used to determine OPC myelination. ⁇ -actin expression was used as an internal control.
  • Figure 11A is an image of a Western blot on cell lysates from CHO-K1 cells expressing S 1P4 FL, S1P4 DN, or control GFP that were treated with SIP to determine active RhoA and total RhoA.
  • FIG. 1 IB is a bar graph showing the relative intensity of active RhoA over total
  • Figure 12 is an image of a Western blot of extracts from oligodendrocyte precursors that were plated into 24-well culture plate in high glucose DME medium with T3/CNTF and treated with 10 ⁇ g/ml anti-SlP4 or control antibody for 72 hours.
  • blocking or inhibiting S 1P4 using antagonists such as a dominant negative mutant, siRNA, or an antibody promotes oligodendrocyte differentiation and myelination, whereas blocking or inhibiting other SIP receptors such as SIP 1 and S1P5 does not have this effect.
  • antagonists of S1P4 can be used therapeutically to treat or prevent diseases where demyelination is involved, such as multiple sclerosis.
  • This disclosure provides an antibody that specifically binds to an epitope within an N-terminal region (residues 23-50) of human S1P4, which promotes
  • oligodendrocyte progenitor cell (OPC) differentiation and myelination thus can be used to treat or prevent demyelinating diseases.
  • OPC oligodendrocyte progenitor cell
  • the term “selectively (or specifically) binds” refers to the ability of an agent (e.g., an antibody or an antigen-binding fragment thereof) to bind to an epitope of a particular protein or peptide of interest (e.g., an N-terminal epitope of human S 1P4 (e.g., an epitope within amino acids 23-50 of SEQ ID NO: 1)) and to not bind in a significant amount to other proteins present in a heterogeneous population of proteins in a sample.
  • an agent e.g., an antibody or an antigen-binding fragment thereof
  • an epitope of a particular protein or peptide of interest e.g., an N-terminal epitope of human S 1P4 (e.g., an epitope within amino acids 23-50 of SEQ ID NO: 1)
  • antibodies raised against human S1P4 can be further selected to obtain those antibodies that are specifically immunoreactive with human S1P4 or a desired portion thereof but not with other proteins (e.g., S1P1, S1P2, S1P3, S1P5).
  • the agent may specifically bind to a particular epitope of a protein without binding in a significant amount to other epitopes of that protein. If an antibody does not significantly bind to a particular epitope, it binds with at least 10-fold, 20-fold, 50-fold, 80-fold, or 100-fold reduced affinity to that epitope as compared to the epitope against which the antibody was raised.
  • an anti-human S1P4 antibody does not significantly bind to human S1P1, S1P2, S1P3, or S1P5 if it binds to the latter proteins with less than 25%, 20%, 10%, 5%, or 1% or less affinity than to the epitope of human S1P4 that the anti-SlP4 antibody was raised against.
  • a first antibody or antigen-binding fragment thereof is said to "compete with” or “specifically compete with” a second antibody if the first antibody competitively inhibits binding of the second antibody to a protein or peptide.
  • the first antibody "competitively inhibits binding" of a second antibody, if the binding of the second antibody is reduced by at least 40%, 50%, 60%, 75%, 80%, 85%, or at least 90%, in the presence of the first antibody, using any of the competition binding assays known in the art (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988)).
  • a “pharmaceutical composition” is a sterile composition of the therapeutic agent (e.g., an antibody or antigen-binding fragment thereof) formulated with a pharmaceutically acceptable carrier, which can be safely administered to a patient.
  • the therapeutic agent e.g., an antibody or antigen-binding fragment thereof
  • a pharmaceutically acceptable carrier which can be safely administered to a patient.
  • an antibody composition is sterile does not make it a pharmaceutical composition; the composition must also not cause undesirable side effects when administered to a patient that outweigh the beneficial effects.
  • the term "monoclonal antibody preparation” refers to a preparation of antibody molecules that contain only one molecular species of antibody molecule.
  • the complementarity determining regions (CDRs) of the antibodies within the monoclonal antibody preparation are identical in all the molecules of the population.
  • the antibodies of the monoclonal antibody preparation thus contain an antigen binding site capable of
  • a "demyelinating disease or disorder” is any disease or disorder of the nervous system in which the myelin sheath of neurons is damaged.
  • demyelinating diseases or disorders of the central nervous system include multiple sclerosis, Devic's disease, inflammatory demyelinating diseases, CNS neuropathies (e.g., those produced by Vitamin B 12 deficiency), central pontine myelinolysis, myelopathies (e.g., Tabes dorsalis); leukoencephalopathies (e.g., progressive multifocal leukoencephalopathy), and
  • demyelinating diseases or disorders of the peripheral nervous system include Guillain-Barre syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, Charcot- Marie-Tooth Disease, copper deficiency, and progressive inflammatory neuropathy.
  • treat refers to any amelioration of symptoms of the disease, improvement in patient survival, or the reversal of the disease.
  • treating can include reduction of demyelination, increase in remyelination, and/or increase in oligodendrocyte differentiation.
  • an “effective amount” or a “therapeutically effective amount” is an amount of the agent (e.g., an antibody or antigen-binding fragment thereof) which alleviates, abates, reduces, or prevents the severity of symptoms of the disease being treated (e.g., multiple sclerosis).
  • a “human antibody” is an antibody whose amino acid sequence is identical or substantially identical to an antibody isolated or obtained from a human cell or human cell line. The antibody is still considered a "human antibody” even if the framework and/or CDRs of the heavy chain variable domain or light chain variable domain of the antibody isolated or obtained from a human cell or human cell line are mutated (e.g., by amino acid
  • the human antibody isolated or obtained from a human cell or human cell line after the human antibody isolated or obtained from a human cell or human cell line is mutated it has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of the antibody isolated or obtained from a human cell or human cell line.
  • six, five, four, three, two, or one amino acid substitutions are made in one, two, three, four, five, and/ or six of the CDRs. In some embodiments, six, five, four, three, two, or one amino acid substitutions are made in one, two, three, or four framework regions of the heavy and/or light chain variable region of the antibody.
  • SIP Sphingosine-1 -phosphate
  • SIP is a sphingolipid that has been shown to mediate a variety of fundamental biological processes such as cell proliferation, migration, invasion, angiogenesis, vascular maturation, and lymphocyte trafficking.
  • This blood borne lipid mediator has the ability to act as an intracellular second messenger as well as an extracellular stimulus through specific G protein-coupled receptors (GPCRs).
  • GPCRs G protein-coupled receptors
  • the intracellular level of SIP is tightly regulated between its synthesis by sphingosine kinases (SphKs) and its degradation by SIP
  • SPPs SIP phosphatases
  • SPL S IP lyase
  • SIPl/Edgl SlP2/Edg5, SlP3/Edg3, SlP4/Edg6, and SlP5/Edg8, have been identified.
  • S IP binds to an SIP receptor, it activates downstream signaling pathways, leading to a variety of cellular responses such as proliferation, cell migration, actin cytoskeletal rearrangement, and adherens junction assembly.
  • S 1P4 Sphingosine-1 -phosphate receptor 4
  • a naturally occurring variant of human S1P4 has an R ⁇ L substitution at amino acid position 365 of SEQ ID NO: l.
  • the topological domains of S1P4 are provided below:
  • the amino acid sequence of the 384 amino acid chimpanzee S1P4 protein (UniProtKB/TrEMBL Database Entry No. H2QEY2) is provided below.
  • the amino acid sequence of the 386 amino acid murine S1P4 protein (UniProtKB/Swioss-Prot Database Entry No. Q9Z0L1) is provided below.
  • S1P4 binds S IP with about a 150-fold lower affinity than other SIP receptors.
  • S 1P4 is coupled to the Gi/o and G12/13 hetrotrimeric G proteins.
  • This disclosure includes antibodies and antigen-binding fragments that specifically bind to S1P4.
  • the antibodies or antigen-binding fragments thereof specifically bind to an epitope within the N-terminal extracellular domain of human S 1P4 (i.e., amino acids 1-50 of SEQ ID NO: l).
  • the antibodies or antigen-binding fragments thereof specifically bind to an epitope within amino acids 23-50 of SEQ ID NO: 1 (i.e., RLIVLHYN HSGRLAGRGG PEDGGLGALR (SEQ ID NO:4)) .
  • the antibodies or antigen-binding fragments thereof have a greater affinity for human S1P4 by at least 100-fold, by at least 50-fold, by at least 10-fold, by at least 5 -fold, or by at least 2-fold, compared with the affinity of the antibodies or antigen- binding fragments thereof for human S 1P1, S 1P2, S1P3, or S1P5.
  • One exemplary anti-S lP4 human antibody that binds an epitope within amino acids 23-50 of SEQ ID NO: l is designated 1F1 1.
  • CDR-Hl heavy chain CDRl
  • CDR2 CDR- H2
  • CDR3 CDR3
  • CDR-Hl heavy chain CDRl
  • CDR2 CDR-H2
  • CDR3 CDR3
  • the nucleic acid sequence of the light chain variable region of the 1F1 1 antibody is provided below with the coding sequences of light chain CDRl (CDR-Ll), CDR2 (CDR-L2), and CDR3 (CDR-L3) based on the Chothia definition, respectively, underlined:
  • CDR-Ll light chain variable region
  • CDR2 CDR-L2
  • CDR3 CDR3
  • CDR-H1, CDR-H2, CDR-H3, CDR- Ll, CDR-L2, CDR-L3, and the four heavy chain variable region framework regions (VH FR1, VH FR2, VH FR3, and VH FR4), and the four light chain variable region framework regions (VL FR1, VL FR2, VL FR3, and VL FR4) are listed in the table below.
  • the CDRs are based upon the Chothia numbering system.
  • VH FR1 CAGGTCAACTTAAGGGAGTCTGGGGGAGGCTTG QVNLRESGGGLVRPGGSLRLS
  • VL FR1 TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCT SSELTQDPAVSVALGQTVRIT
  • VL FR3 GGGGTCCCTGATCGGTTCTCTGGCTCCATCGAC GVPDRFSGSIDSSSNSASLTI
  • VL FR4 TTCGGCGGAGGGACCCAGCTCACCGTTTTA FGGGTQLTVL
  • This disclosure provides an antibody or the antigen binding fragment thereof that specifically binds S1P4 and comprises: (i) a CDR-Hl comprising an amino acid sequence set forth in SEQ ID NOs.: 11, 50, 51, or 52 with three, two, one, or no amino acid substitutions; (ii) a CDR-H2 comprising an amino acid sequence set forth in SEQ ID NOs.: 13, 53, 54, or 55 with three, two, one, or no amino acid substitutions; and (iii) a CDR-H3 comprising an amino acid sequence set forth in SEQ ID NOs.: 15 or 56 with three, two, one, or no amino acid substitutions. In some embodiments, the amino acid substitutions are conservative.
  • this antibody or the antigen binding fragment thereof specifically binds S1P4 on the surface of a cell. In another embodiment, this antibody or the antigen binding fragment thereof specifically binds non-denatured S1P4. In some embodiments, this antibody or the antigen binding fragment thereof further comprises: i) a CDR-L1 comprising an amino acid sequence set forth in SEQ ID NOs.: 17 or 57 with three, two, one, or no amino acid substitutions; (ii) a CDR-L2 comprising an amino acid sequence set forth in SEQ ID NOs.: 19 or 58 with three, two, one, or no amino acid substitutions; and (iii) a CDR-L3 comprising an amino acid sequence set forth in SEQ ID NOs.: 21 or 59 with three, two, one, or no amino acid substitutions.
  • the amino acid substitutions are conservative.
  • this disclosure provides an antibody or the antigen binding fragment thereof specifically binds S 1P4 and comprises (i) a CDR-H1 comprising an amino acid sequence set forth in SEQ ID NOs.: 1 1, 50, 51, or 52; (ii) a CDR-H2 comprising an amino acid sequence set forth in SEQ ID NOs.: 13, 53, 54, or 55; and (iii) a CDR-H3 comprising an amino acid sequence set forth in SEQ ID NOs.: 15 or 56.
  • this antibody or the antigen binding fragment thereof further comprises: i) a CDR-L1 comprising an amino acid sequence set forth in SEQ ID NOs.: 17 or 57; (ii) a CDR- L2 comprising an amino acid sequence set forth in SEQ ID Nos.: 19 or 58; and (iii) a CDR- L3 comprising an amino acid sequence set forth in SEQ ID NOs.: 21 or 59.
  • the antibody is a full-length human antibody.
  • the nucleic acid sequence of the 1F1 1 IgG2 heavy chain is provided below with the heavy chain variable region underlined:
  • the amino acid sequence of the 1F1 1 IgG2 heavy chain is provided below with the heavy chain variable region underlined: QVNLRESGGGLVRPGGSLRLSCAASGFTVSGYAMSWVRQAPGKGLEWVSAISRFSDTTYSADSVKGRF TISRDNSKNTVYLQMNSLRAEDSAVYYCARDPSGHGMDVWGQGTLVTVSAASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD HKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD
  • amino acid sequence of the 1F1 1 lambda light chain is shown below with the light chain variable region underlined:
  • This disclosure also includes antibodies or antigen-binding fragments thereof that specifically bind human S1P4 and that have heavy chain variable regions that are: 85%, 86%,
  • these antibodies or antigen binding fragments inhibit SIP binding to human S1P4; inhibit signaling through
  • these antibodies or antigen-binding fragments thereof have an EC50 of 0.2 nM, 0.3 nM, 0.4 nM, 0.5nM, 0.6 nM, 0.7 nM, 0.8, or 0.9 nM. In some embodiments, these antibodies further include a light chain variable region that is: 85%, 86%, 87%, 88%, 89%,
  • these antibodies or antigen binding fragments inhibit S IP binding to human S1P4; inhibit signaling through S1P4; promote oligodendrocyte precursor cell (OPC) differentiation into myelinating
  • these antibodies or antigen-binding fragments thereof have an EC50 of 0.2 nM, 0.3 nM, 0.4 nM, 0.5nM, 0.6 nM, 0.7 nM, 0.8, or 0.9 nM.
  • This disclosure also includes antibodies or antigen-binding fragments that specifically bind S1P4 and that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or all four of the framework regions of the heavy chain comprising the amino acid sequences set forth in SEQ ID NOs: 23, 25, 27, and 29, and/or four or fewer (e.g., four, three or fewer, two or fewer, or one) amino acid substitutions in one, two, or all three CDRs, of the heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 11, 13, and 15.
  • the application also includes antibodies that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or all four of the framework regions of the light chain comprising the amino acid sequences set forth in SEQ ID NOs: 31, 33, 35, and 37, and/or four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, or all three CDRs, of the light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 17, 19, and 21.
  • the antibodies of this disclosure include antibodies that specifically bind human S1P4 and that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or four of the framework regions of the heavy chain comprising the amino acid sequences set forth in SEQ ID NOs.: 23, 25, 27, and 29, and/or four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, or three CDRs, of the heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: l 1, 13, and 15; and four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or four of the framework regions of the light chain comprising the amino acid sequences set forth in SEQ ID NOs.: 31, 33, 35, and 37, and/or four or fewer
  • S1P4 also include polypeptides comprising a heavy chain variable region that comprises
  • CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs.:
  • these antibodies or antigen-binding fragments are 11, 13, and 15. In certain embodiments, these antibodies or antigen-binding fragments
  • polypeptides comprising a light chain variable region that comprises
  • CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs.:
  • the VH and or VL region can be linked to a constant region (e.g., a wild-type human Fc region or an Fc region that includes one or more alterations).
  • the constant region comprises a CHI domain and a hinge region.
  • the constant region comprises a CH3 domain.
  • the VL of the antibody is linked to a human kappa or lambda light chain constant region.
  • the antibody has an isotype selected from the group consisting of IgG (e.g., IgGl, IgG2, IgG3, IgG4), Ig A (e.g., IgAl, IgA2), IgD, IgE, and IgM.
  • the antibody has an IgG2 isotype.
  • the constant region can be a human Fc region, e.g., a wild-type Fc region, or an Fc region that includes one or more amino acid substitutions.
  • the constant region can have substitutions that modify the properties of the antibody (e.g., increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
  • the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237 (based on Kabat numbering).
  • Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation.
  • antibodies may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260.
  • Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30: 105-08). See also, e.g., U.S. 2005-0037000.
  • the antibodies or antigen binding fragments thereof can be conjugated to one or more agents that are useful for treating or preventing the condition the antibody or antigen binding fragment thereof is being used to treat or prevent (e.g., a demyelinating disease).
  • agents can be, e.g., miRNAs, miRNA mimics, siRNAs, anti-miRs, antisense nucleic acids, ribozymes, small molecule compounds, and other chemical moieties.
  • Such antibodies or antigen binding fragments can be used e.g., to deliver the bound agent(s) to a cell or tissue of interest that expresses S1P4.
  • Antibodies can be selected for use based on improved potency, higher affinity or avidity for S1P4, and/or reduced immunogenicity than previously known S1P4 antibodies. Methods of determining potency, affinity or avidity, and immunogenicity of antibodies are within the skill of the ordinary artisan.
  • Antibodies such as those described above, can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences. Methods of generating variants (e.g., comprising amino acid substitutions) of any of the anti-SlP4 antibodies are well known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a prepared DNA molecule encoding the antibody or any portion thereof (e.g., a framework region, a CDR, a constant region).
  • PCR mutagenesis is well known in the art (see, e.g., Carter et al, Nucleic Acids Res., 13 :4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987)).
  • PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990); and Vallette et al, Nuc. Acids Res. 17:723-733 (1989).
  • Another method for preparing sequence variants, cassette mutagenesis is based on the technique described by Wells et al, Gene, 34:315-323 (1985).
  • an anti-SlP4 antibody or antigen-binding fragment thereof described herein is modified, e.g., by mutagenesis, to provide a pool of modified antibodies.
  • the modified antibodies are then evaluated to identify one or more antibodies having altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased stability in vivo).
  • display library technology is used to select or screen the pool of modified antibodies.
  • Higher affinity antibodies are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used.
  • the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particularly within 10, 5, or 3 amino acids of a CDR junction. In the case of antibodies, mutagenesis can also be limited to one or a few of the CDRs, e.g., to make step-wise improvements.
  • mutagenesis is used to make an antibody more similar to one or more germline sequences.
  • One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.
  • mutagenesis is used to substitute or insert one or more germline residues into a CDR region.
  • the germline CDR residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified.
  • activity e.g., binding or other functional activity
  • Similar mutagenesis can be performed in the framework regions.
  • a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody.
  • the selection can be performed using at least 2, 3, 5, or 10 germline sequences.
  • identifying a similar germline sequence can include selecting one such sequence.
  • identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations, more than one or two germline sequences are used, e.g., to form a consensus sequence.
  • sequence identity between two sequences are performed as follows.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the antibody may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern).
  • altered means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody.
  • an antibody has CDR sequences (e.g., a Chothia or Kabat CDR) that differ from those of SEQ ID NOs: 11, 13, 15, 17, 19, and 21.
  • CDR sequences that differ from those of the 1F1 1 antibody described herein include amino acid changes, such as substitutions of 1, 2, 3, or 4 amino acids if a CDR is 5-7 amino acids in length, or substitutions of 1, 2, 3, 4, or 5 of amino acids in the sequence of a CDR if a CDR is 8 amino acids or greater in length.
  • the amino acid that is substituted can have similar charge, hydrophobicity, or stereochemical characteristics. In some embodiments, the amino acid substitution(s) is a conservative substitution.
  • the amino acid substitution(s) is a non-conservative substitution. Such substitutions are within the ordinary skill of an artisan.
  • the antibody or antibody fragments thereof that contain the substituted CDRs can be screened to identify antibodies having one or more of the features described herein (e.g., specifically binding to human S1P4, inhibiting the binding of S IP to human S1P4; promote differentiation of OPCs to myelinating oligodendrocytes; increase expression of MBP and/or MOG in oligodendrocytes).
  • FRs structure framework regions
  • Changes to FRs include, but are not limited to, humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al, J. Immun., 147:2657-62 (1991); Morgan et al, Immunology, 86:319-24 (1995)), or changing the species from which the constant region is derived.
  • the anti-SlP4 antibodies can be in the form of full length antibodies, or in the form of low molecular weight forms (e.g., biologically active antibody fragments or minibodies) of the anti-SlP4 antibodies, e.g., Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, sc(Fv)2, and
  • anti-SlP4 antibodies encompassed by this disclosure include single domain antibody (sdAb) containing a single variable chain such as, VH or VL, or a biologically active fragment thereof.
  • sdAb single domain antibody
  • sdAb Like a whole antibody, a sdAb is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, sdAbs are much smaller than common antibodies and even smaller than Fab fragments and single-chain variable fragments.
  • an anti-SlP4 antibody or antigen-binding fragment thereof or low molecular weight antibodies thereof specifically binds to human S1P4, inhibits the binding of SIP to human S1P4, promotes oligodendrocyte precursor cell (OPC)
  • Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb, and Fv) of human SlP4-binding antibodies may be prepared by proteolytic digestion of intact anti-SlP4 antibodies.
  • antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab')2 or Fab'; and plasmin digestion of whole antibodies yields Facb fragments.
  • antibody fragments can be produced recombinantly.
  • nucleic acids encoding the antibody fragments of interest can be constructed, introduced into an expression vector, and expressed in suitable host cells. See, e.g., Co, M.S. et al., J.
  • Antibody fragments can be isolated from the antibody phage libraries.
  • Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al, Bio/Technology, 10: 163-167 (1992)).
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.
  • Minibodies of anti-SlP4 antibodies include diabodies, single chain (scFv), and single- chain (Fv)2 (sc(Fv)2).
  • a “diabody” is a bivalent minibody constructed by gene fusion (see, e.g., Holliger, P. et al, Proc. Natl. Acad. Sci. U. S. A., 90:6444-6448 (1993); EP 404,097; WO 93/1 1161).
  • Diabodies are dimers composed of two polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers.
  • the number of amino acid residues that constitute a linker can be between 2 to 12 residues (e.g., 3-10 residues or five or about five residues).
  • the linkers of the polypeptides in a diabody are typically too short to allow the VL and VH to bind to each other.
  • the VL and VH encoded in the same polypeptide chain cannot form a single-chain variable region fragment, but instead form a dimer with a different single-chain variable region fragment.
  • a diabody has two antigen- binding sites.
  • An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see e.g., Huston et al, Proc. Natl. Acad. Sci. U. S. A., 85:5879-5883 (1988); and Pluckthun, "The Pharmacology of Monoclonal Antibodies” Vol.113, Ed Resenburg and Moore, Springer Verlag, New York, pp.269-315, (1994)).
  • the order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: [VH] linker [VL]; or [VL] linker [VH].
  • the H chain V region and L chain V region in an scFv may be derived from any anti-S lP4 antibody or antigen-binding fragment thereof described herein.
  • An sc(Fv)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson, et al, J. Immunol. Methods, (1999) 231 : 177-189 (1999)).
  • An sc(Fv)2 can be prepared, for example, by connecting scFvs with a linker.
  • the sc(Fv)2 of the present invention include antibodies preferably in which two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]), beginning from the N terminus of a single-chain polypeptide; however the order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order. Examples of arrangements are listed below:
  • the linker is a peptide linker. Any arbitrary single-chain peptide comprising about three to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18) can be used as a linker.
  • peptide linkers include: Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO:42); Ser Gly Gly Gly (SEQ ID NO:43); Gly Gly Gly Ser (SEQ ID NO:44); Ser Gly Gly Gly Gly (SEQ ID NO:45); Gly Gly Gly Gly Gly Ser (SEQ ID NO:46); Ser Gly Gly Gly Gly Gly (SEQ ID NO:47); Gly Gly Gly Gly Gly Ser (SEQ ID NO:48); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO:49); (Gly Gly Gly Ser (SEQ ID NO:44) n , wherein n is an integer of one or more; and (Ser Gly Gly Gly (SEQ ID NO:45) n , wherein n is an integer of one or more.
  • the linker is a synthetic compound linker (chemical cross- linking agent).
  • cross-linking agents that are available on the market include N- hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol
  • disulfosuccinimidyl tartrate sulfo-DST
  • bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone BSOCOES
  • bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone sulfo-BSOCOES
  • the amino acid sequence of the heavy chain variable region (VH) or the light chain variable region (VL) in the minibodies may include modifications such as substitutions, deletions, additions, and/or insertions.
  • the modification may be in one or more of the CDRs of the anti-SlP4 antibody or antigen-binding fragment thereof.
  • the modification involves one, two, or three amino acid substitutions in one or more CDRs and/or framework regions of the VH and/or VL domain of the anti- human S1P4 minibody. Such substitutions are made to improve the binding, functional activity and/or reduce immunogenicity of the anti-SlP4 minibody.
  • the substitutions are conservative amino acid substitutions.
  • one, two, or three amino acids of the CDRs of the anti-human S 1P4 antibody or antigen-binding fragment thereof may be deleted or added as long as there is S1P4 binding and/or functional activity when VH and VL are associated.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of the human S1P4 protein.
  • Other such antibodies may combine a S1P4 binding site with a binding site for another protein (e.g., Sp35/LLNGO-l/LRRN6 (see, e.g., WO2008/086006); DR-6 (see, e.g., WO 2010/062904); and T-cell antigens (e.g., CD2, CD3, CD5, CD6, CD7, TCR)).
  • Bispecific antibodies can be prepared as full length antibodies or low molecular weight forms thereof (e.g., F(ab') 2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).
  • Traditional production of full length bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the proportions of the three polypeptide fragments. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields.
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the Cm domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods.
  • the "diabody” technology provides an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies describe herein can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • An exemplary dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • a multivalent antibody can comprise (or consist of) three to about eight (e.g., four) antigen binding sites.
  • the multivalent antibody optionally comprises at least one polypeptide chain (e.g., at least two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VDl-(Xl) n -VD2-(X2) n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or peptide spacer, and n is 0 or 1.
  • the antibodies disclosed herein may be conjugated antibodies which are bound to various molecules including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, cytotoxic agents, and drugs.
  • macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, cytotoxic agents, and drugs.
  • an anti-S lP4 antibody or antigen-binding fragment thereof are modified with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold.
  • the anti-S lP4 antibody or antigen-binding fragment thereof can be associated with (e.g., conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight.
  • Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
  • the anti-SlP4 antibody or antigen-binding fragment thereof can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone.
  • a water soluble polymer e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone.
  • examples of such polymers include polyalkylene oxide
  • polyethylene glycol PEG
  • polypropylene glycols polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched
  • the antibodies or antigen-binding fragments thereof can also be conjugated to siRNAs, miRNAs, or anti-miRs to deliver the siRNA, miRNA, or anti-miR to cells expressing S 1P4 (see, e.g., Song et al, Nat. Biotechnol., 23(6):709-17 (2005); Schneider et al, Molecular Therapy Nucleic Acids, l :e46 (2012)).
  • the siRNAs, miRNAs, or anti-miRs can target S1P4 or components of the S1P4- signaling pathway.
  • the siRNAs, miRNAs, or anti-miRs can target genes involved in the disease being treated (e.g., a demyelinating disease).
  • the antibodies or antigen-binding fragments thereof can also be conjugated to small molecules and other chemical moieties.
  • conjugated antibodies can be prepared by performing chemical modifications on the antibodies or the lower molecular weight forms thereof described herein. Methods for modifying antibodies are well known in the art (e.g., US 5057313 and US 5156840).
  • the efficacy of a therapeutic antibody can be improved by increasing its serum persistence, thereby allowing higher circulating levels, less frequent administration, and reduced doses.
  • the half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn.
  • FcRn which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.
  • the antibodies of the present disclosure have one or more mutations at the interface between the CH2 and CH3 domains, such as T250Q/M428L and M252Y/S254T/T256E in combination with H433K/N434F (the numbering is according to the EU index), which increase the binding affinity to FcRn and the half-life of IgG 1 in vivo.
  • the antibodies herein have a modified Fc region comprising at least one modification relative to a wild-type human Fc region, where the modification is selected from the group consisting of 434S, 252Y/428L, 252Y/434S, and 428L/434S (the numbering is according to the EU index).
  • the anti-human S 1P4 antibodies (or antibody binding fragments thereof) of this disclosure may be produced in bacterial or eukaryotic cells.
  • Some antibodies, e.g., Fab's can be produced in bacterial cells, e.g., E. coli cells.
  • Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, 293T, COS, NIH3T3, HeLa).
  • antibodies e.g., scFv's
  • a yeast cell such as Pichia (see, e.g., Powers et al, J Immunol Methods. 251 : 123-35 (2001)), Hanseula, or
  • a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in suitable host cells. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.
  • the expression vector should have characteristics that permit amplification of the vector in the bacterial cells.
  • E. coli such as JM109, DH5a, HB101, or XLl-Blue
  • the vector must have a promoter, for example, a lacZ promoter (Ward et al, 341 :544-546 (1989), araB promoter (Better et al, Science, 240: 1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli.
  • a promoter for example, a lacZ promoter (Ward et al, 341 :544-546 (1989), araB promoter (Better et al, Science, 240: 1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli.
  • Such vectors include, for example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-l (Pharmacia), "QIAexpress system” (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 R A polymerase).
  • the expression vector may contain a signal sequence for antibody secretion.
  • the pelB signal sequence Lei et al, J. Bacteriol, 169:4379 (1987)
  • calcium chloride methods or electroporation methods may be used to introduce the expression vector into the bacterial cell.
  • the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al, Nature, 277: 108 (1979)), MMLV- LTR promoter, EF la promoter (Mizushima et al. , Nucleic Acids Res. , 18:5322 (1990)), or CMV promoter.
  • SV40 promoter Mulligan et al, Nature, 277: 108 (1979)
  • MMLV- LTR promoter MMLV- LTR promoter
  • EF la promoter EF la promoter
  • CMV promoter CMV promoter
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5, 179,017).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.
  • vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
  • antibodies are produced in mammalian cells.
  • exemplary mammalian host cells for expressing an antibody include Chinese Hamster Ovary (CHO cells) (including dhfr ⁇ CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Set USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.
  • human embryonic kidney 293 cells e.g., 293, 293E, 293T
  • COS cells e.g., NIH3T3 cells
  • lymphocytic cell lines e.g., NS0 myeloma cells and SP2 cells
  • a cell from a transgenic animal e.g., a transgenic mammal.
  • the cell is a mammary epithelial cell.
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an anti-SlP4 antibody is introduced into dhfr CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and the antibody is recovered from the culture medium.
  • Antibodies can also be produced by a transgenic animal.
  • U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest.
  • the antibody can be purified from the milk, or for some applications, used directly. Animals are also provided comprising one or more of the nucleic acids described herein.
  • Antibodies and antigen-binding fragments can also be obtained using display libraries such as a phage display library.
  • display libraries such as a phage display library.
  • an extracellular domain of S1P4 e.g., the N- terminal amino acids 23-50 of SEQ ID NO: 1
  • the display library can display a polypeptide that includes a VH domain and a VL domain, an Fab fragment, a single chain Fv, or any other format.
  • Antibody libraries can be constructed by a number of methods (see, e.g., de Haard et al, 1999, J. Biol. Chem. 274: 18218-30; Hoogenboom et al, 1998, Immunotechnology 4: 1- 20; Hoogenboom et al, 2000, Immunol. Today 21 :371-378, and Hoet et al. (2005) Nat.
  • the methods can be implemented such that variation is introduced into a single immunoglobulin domain (e.g., VH or VL) or into multiple immunoglobulin domains (e.g., VH and VL).
  • the variation can be introduced into an immunoglobulin variable domain, e.g., in the region of one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4, referring to such regions of either and both of heavy and light chain variable domains.
  • the variation(s) may be introduced into all three CDRs of a given variable domain, or into CDR1 and CDR2, e.g., of a heavy chain variable domain.
  • antibody libraries are constructed by inserting diverse oligonucleotides that encode CDRs into the corresponding regions of the nucleic acid.
  • the oligonucleotides can be synthesized using monomeric nucleotides or trinucleotides. For example, Knappik et al., 2000, J. Mol. Biol. 296:57-86 describe a method for constructing CDR encoding
  • oligonucleotides using trinucleotide synthesis and a template with engineered restriction sites for accepting the oligonucleotides.
  • an animal e.g., a rodent
  • the animal is optionally boosted with the antigen to further stimulate the response.
  • spleen cells are isolated from the animal, and nucleic acid encoding VH and/or VL domains is amplified and cloned for expression in the display library.
  • antibody libraries are constructed from nucleic acid amplified from naive germline immunoglobulin genes.
  • the amplified nucleic acid includes nucleic acid encoding the VH and/or VL domain.
  • Amplification can include PCR, e.g., with primers that anneal to the conserved constant region, or another amplification method.
  • the antibody or fragment is typically covalently linked to a bacteriophage coat protein.
  • the linkage results from translation of a nucleic acid encoding the protein component fused to the coat protein.
  • the linkage can include a flexible peptide linker, a protease site, or an amino acid incorporated as a result of suppression of a stop codon.
  • Bacteriophage displaying the protein component can be grown and harvested using standard phage preparatory methods, e.g. PEG precipitation from growth media.
  • the nucleic acid encoding the selected protein components can be isolated from cells infected with the selected phages or from the phage themselves, after amplification. Individual colonies or plaques can be picked, the nucleic acid isolated and sequenced.
  • the antibody can be optimized (e.g., to improve binding, reduce immunogenicity, increase half-life, etc.) by mutation(s) and additional rounds of selection using the display technology. Phage display is described, for example, in U.S. Pat. No.
  • the antibodies of the present disclosure can be isolated from inside or outside (such as medium) of the host cell and purified as substantially pure and homogenous antibodies. Methods for isolation and purification commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel
  • Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption
  • Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC.
  • Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences).
  • the present disclosure also includes antibodies that are highly purified using these purification methods. Characterization of the Antibodies
  • the SlP4-binding properties of the antibodies described herein may be measured by any standard method, e.g., one or more of the following methods: OCTET ® , Surface Plasmon Resonance (SPR), BIACORETM analysis, Enzyme Linked Immunosorbent Assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and Fluorescence Resonance Energy Transfer (FRET).
  • OCTET ® Surface Plasmon Resonance
  • ELISA Enzyme Linked Immunosorbent Assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FRET Fluorescence Resonance Energy Transfer
  • the binding interaction of a protein of interest can be analyzed using the OCTET ® systems.
  • OCTET ® QK e and QK are used to determine protein interactions, binding specificity, and epitope mapping.
  • the OCTET ® systems provide an easy way to monitor real-time binding by measuring the changes in polarized light that travels down a custom tip and then back to a sensor.
  • SPR Surface Plasmon Resonance
  • BIA Biomolecular Interaction Analysis
  • Epitopes can also be directly mapped by assessing the ability of different antibodies to compete with each other for binding to human S1P4 using BIACORE chromatographic techniques (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160: 191-198).
  • an enzyme immunoassay When employing an enzyme immunoassay, a sample containing an antibody, for example, a culture supernatant of antibody -producing cells or a purified antibody is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, and after washing, an enzyme substrate such as p-nitrophenylphosphate is added, and the absorbance is measured to evaluate the antigen binding activity.
  • an enzyme substrate such as p-nitrophenylphosphate
  • Immune-mediated effector functions include two major mechanisms: antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Both of them are mediated by the constant region of the immunoglobulin protein.
  • the antibody Fc domain is, therefore, the portion that defines interactions with immune effector mechanisms.
  • IgG antibodies activate effector pathways of the immune system by binding to members of the family of cell surface Fey receptors and to Clq of the complement system. Ligation of effector proteins by clustered antibodies triggers a variety of responses, including release of inflammatory cytokines, regulation of antigen production, endocytosis, and cell killing. In some clinical applications these responses are crucial for the efficacy of a monoclonal antibody. In others they provoke unwanted side effects such as inflammation and the elimination of antigen-bearing cells.
  • the present disclosure provides anti-human S 1P4 antibodies with reduced effector functions.
  • Effector function of an anti-SlP4 antibody of the present invention may be determined using one of many known assays.
  • the anti-SlP4 antibody's effector function may be increased or reduced relative to a second anti-S lP4 antibody.
  • the second anti-SlP4 antibody may be any antibody that binds S1P4 specifically.
  • the second SlP4-specific antibody may be any of the antibodies of the invention, such as the antibody designated 1F1 1.
  • the second anti-S lP4 antibody may be the unmodified or parental version of the antibody.
  • Effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC), whereby antibodies bind Fc receptors on cytotoxic T cells, natural killer (NK) cells, or macrophages leading to cell death, and complement-dependent cytotoxicity (CDC), which is cell death induced via activation of the complement cascade (reviewed in Daeron, Annu. Rev. Immunol, 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol, 2:77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991)).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using standard assays that are known in the art (see, e.g., WO 05/018572, WO 05/003175, and U.S. 6,242,195).
  • FcRs Fc receptors
  • the affinity of an antibody for a particular FcR, and hence the effector activity mediated by the antibody, may be modulated by altering the amino acid sequence and/or post-translational modifications of the Fc and/or constant region of the antibody.
  • FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FceR, for IgA as FcaR and so on.
  • FcyR FcyRI
  • FcyRIII CD16
  • Both FcyRII and FcyRIII have two types: FcyRIIA (CD32) and FcyRIIB (CD32); and FcyRIIIA (CD 16a) and FcyRIIIB (CD 16b).
  • FcyRII (CD32) includes the isoforms Ila, Ilbl, IIb2 IIb3, and lie.
  • the anti-SlP4 antibodies of the present invention include modifications of one or more of the aforementioned residues to decrease effector function.
  • Effector functions can also be avoided by using antibody fragments lacking the Fc domain such as Fab, Fab'2, or single chain Fv.
  • An alternative is to use the IgG4 subtype antibody, which binds to FcyRI but which binds poorly to Clq and FcyRII and RIII.
  • the IgG2 subtype also has reduced binding to Fc receptors, but retains significant binding to the HI 31 allotype of FcyRIIa and to Clq. Thus, additional changes in the Fc sequence are required to eliminate binding to all the Fc receptors and to Clq.
  • Another approach for reducing antibody effector function is by mutating amino acids on the surface of the antibody that are involved in effector binding interactions (Lund, J., et al. (1991) J. Immunol. 147(8): 2657-62; Shields, R. L. et al. (2001) J. Biol. Chem. 276(9): 6591-604).
  • sites of N-linked glycosylation can be removed as a means of reducing effector function.
  • a large number of Fc variants having altered and/or reduced affinities for some or all Fc receptor subtypes (and thus for effector functions) are known in the art.
  • Non-limiting examples of substitutions that reduce effector function include one or more of: K322A; L234A/L235A; G236T; G236R; G236Q; H268A; H268Q; V309L; A330S;P331 S; V234A/G237A/P238S/ H268A/V309L/A330S/P331S;
  • Anti-SlP4 antibodies of the present invention with reduced effector function include antibodies with reduced binding affinity for one or more Fc receptors (FcRs) relative to a parent (e.g., IF 11 IgG2) or non-variant anti-human S1P4 antibody.
  • FcRs Fc receptors
  • anti-SlP4 antibodies with reduced FcR binding affinity includes anti-human S1P4 antibodies that exhibit a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or higher decrease in binding affinity to one or more Fc receptors compared to a parent or non-variant anti-human S 1P4 antibody.
  • an anti-human S 1P4 antibody with reduced effector function binds to an FcR with about 10-fold less affinity relative to a parent (e.g., 1F 11 IgG2) or non-variant antibody.
  • an anti-SlP4 antibody with reduced effector function binds to an FcR with about 15-fold less affinity or with about 20-fold less affinity relative to a parent (e.g., 1F1 1 IgG2) or non-variant antibody.
  • the FcR receptor may be one or more of FcyRI (CD64), FcyRII (CD32), and FcyRIII, and isoforms thereof, and FceR, Fc ⁇ R, Fc5R, and/or an FcaR.
  • an anti-SlP4 antibody with reduced effector function exhibits a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or higher decrease in binding affinity to FcyRIIa.
  • the antibody-antigen complex binds complement, resulting in the activation of the complement cascade and generation of the membrane attack complex.
  • Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen; thus the activation of the complement cascade is regulated in part by the binding affinity of the immunoglobulin to Clq protein.
  • Clq first component of the complement system
  • residues of the IgG molecule are involved in binding to Clq including the Glu318, Lys320 and Lys322 residues on the CH2 domain, amino acid residue 331 located on a turn in close proximity to the same beta strand, the Lys235 and Gly237 residues located in the lower hinge region, and residues 231 to 238 located in the N-terminal region of the CH2 domain (see e.g., Xu et al, J. Immunol. 150: 152A (Abstract) (1993),W094/29351; Tao et al, J. Exp. Med., 178:661-667 (1993); Brekke et al, Eur. J.
  • Anti-SlP4 antibodies with reduced Clq binding can comprise an amino acid substitution at one, two, three, or four of amino acid positions 270, 322, 329 and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat.
  • IgGl two mutations in the COOH terminal region of the CH2 domain of human IgGl— K322A and P329A— do not activate the CDC pathway and were shown to result in more than a 100 fold decrease in Clq binding (US 6,242, 195).
  • an anti-human S1P4 antibody of the present invention exhibits reduced binding to a complement protein relative to a second anti-human S1P4 antibody.
  • an anti-S lP4 antibody of the invention exhibits reduced binding to Clq by a factor of about 1.5-fold or more, about 2-fold or more, about 3- fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, or about 15-fold or more, relative to a second anti-SlP4 antibody.
  • one or more of these residues may be modified, substituted, or removed or one or more amino acid residues may be inserted so as to decrease CDC activity of the anti-human S1P4 antibodies provided herein.
  • a native sequence Fc or constant region comprises an amino acid sequence identical to the amino acid sequence of a Fc or constant chain region found in nature.
  • a control molecule used to assess relative effector function comprises the same type/subtype Fc region as does the test or variant antibody.
  • a variant or altered Fc or constant region comprises an amino acid sequence which differs from that of a native sequence heavy chain region by virtue of at least one amino acid modification (such as, for example, post- translational modification, amino acid substitution, insertion, or deletion).
  • the variant constant region may contain one or more amino acid substitutions, deletions, or insertions that results in altered post-translational modifications, including, for example, an altered glycosylation pattern.
  • a parent antibody or Fc region is, for example, a variant having normal effector function used to construct a constant region (i.e., Fc) having altered, e.g., decreased effector function.
  • Antibodies with reduced effector function(s) may be generated by engineering or producing antibodies with variant constant, Fc, or heavy chain regions.
  • Recombinant DNA technology and/or cell culture and expression conditions may be used to produce antibodies with altered function and/or activity.
  • recombinant DNA technology may be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (such as, for example, Fc or constant regions) that affect antibody function including effector functions.
  • changes in post-translational modifications such as, e.g.
  • glycosylation patterns may be achieved by manipulating the host cell and cell culture and expression conditions by which the antibody is produced.
  • an anti-human S1P4 antibody comprising one or more heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 11, VH CDR2 of SEQ ID NO: 13, and VH CDR3 of SEQ ID NO: 15, wherein the antibody further comprises a variant Fc region that confers reduced effector function compared to a native or parental Fc region.
  • the anti-human S1P4 antibody comprises at least two of the CDRs, and in other embodiments the antibody comprises all three of the heavy chain CDR sequences.
  • an anti-human S1P4 antibody comprising one or more light chain CDR sequences selected from VL CDR1 of SEQ ID NO: 17, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO:21 the antibody further comprising a variant Fc region that confers reduced effector function compared to a native or parental Fc region.
  • the anti-human S1P4 antibody comprises at least two of the light chain CDRs, and in other embodiments the antibody comprises all three of the light chain CDR sequences.
  • the anti-human S1P4 antibody with reduced effector function comprises all three light chain CDR sequences (CDRs 1, 2, and 3) of SEQ ID NO:9 and comprises all three heavy chain CDR sequences (CDRs l, 2, and 3) of SEQ ID NO:7.
  • the anti-human S 1P4 antibody with reduced effector function comprises: three or fewer, two or fewer, or one amino acid substitution in one, two, or three CDRs of SEQ ID NO: 9 and three or fewer, two or fewer, or one amino acid substitution in one, two, or three CDRs of SEQ ID NO:7.
  • the invention relates to an anti-human S1P4 antibody comprising a VL sequence set forth in SEQ ID NO: 9, the antibody further comprising a variant Fc region that confers reduced effector function compared to a native or parental (e.g., 1F1 1 IgG2) Fc region.
  • the invention relates to an anti-humanSlP4 antibody comprising a VH sequence selected from the group consisting of SEQ ID NO: 7, the antibody further comprising a variant Fc region that confers reduced effector function compared to a native or parental (e.g., 1F 11 IgG2) Fc region.
  • Glycan removal produces a structural change that should greatly reduce binding to all members of the Fc receptor family across species.
  • the glycans oligosaccharides
  • the sugar residues making contact with specific amino acid residues on the opposing CH2 domain.
  • Different glycosylation patterns are associated with different biological properties of antibodies (Jefferis and Lund, 1997, Chem. Immunol, 65: 1 11-128; Wright and Morrison, 1997, Trends BiotechnoL, 15: 26-32). Certain specific glycoforms confer potentially advantageous biological properties.
  • Loss of the glycans changes spacing between the domains and increases their mobility relative to each other and is expected to have an inhibitory effect on the binding of all members of the Fc receptor family.
  • in vitro studies with various glycosylated antibodies have demonstrated that removal of the CH2 glycans alters the Fc structure such that antibody binding to Fc receptors and the complement protein C1Q are greatly reduced.
  • Another known approach to reducing effector functions is to inhibit production of or remove the N- linked glycans at position 297 (EU numbering) in the CH2 domain of the Fc (Nose et al, 1983 PNAS 80: 6632; Leatherbarrow et al., 1985 Mol. Immunol. 22: 407; Tao et al, 1989 J. Immunol. 143 : 2595; Lund et al, 1990 Mol. Immunol. 27: 1 145; Dorai et al, 1991
  • the oligosaccharide structure can affect properties relevant to protease resistance, the serum half-life of the antibody mediated by the FcRn receptor, phagocytosis and antibody feedback, in addition to effector functions of the antibody (e.g., binding to the complement complex CI, which induces CDC, and binding to FcyR receptors, which are responsible for modulating the ADCC pathway) (Nose and Wigzell, 1983;
  • another means of modulating effector function of antibodies includes altering glycosylation of the antibody constant region.
  • Altered glycosylation includes, for example, a decrease or increase in the number of glycosylated residues, a change in the pattern or location of glycosylated residues, as well as a change in sugar structure(s).
  • the oligosaccharides found on human IgGs affects their degree of effector function (Raju, T.S. BioProcess International April 2003. 44-53); the microheterogeneity of human IgG oligosaccharides can affect biological functions such as CDC and ADCC, binding to various Fc receptors, and binding to Clq protein (Wright A. & Morrison SL. TIBTECH 1997, 15 26- 32; Shields et al. J Biol Chem. 2001 276(9):6591-604; Shields et al. J Biol Chem. 2002;
  • IgG IgG to bind Clq and activate the complement cascade may depend on the presence, absence or modification of the carbohydrate moiety positioned between the two CH2 domains (which is normally anchored at Asn297) (Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).
  • Glycosylation sites in an Fc-containing polypeptide for example an antibody such as an IgG antibody, may be identified by standard techniques. The identification of the glycosylation site can be experimental or based on sequence analysis or modeling data.
  • Consensus motifs that is, the amino acid sequence recognized by various glycosyl transferases, have been described.
  • the consensus motif for an N-linked glycosylation motif is frequently NXT or NXS, where X can be any amino acid except proline.
  • Several algorithms for locating a potential glycosylation motif have also been described. Accordingly, to identify potential glycosylation sites within an antibody or Fc- containing fragment, the sequence of the antibody is examined, for example, by using publicly available databases such as the website provided by the Center for Biological Sequence Analysis (see NetNGlyc services for predicting N-linked glycosylation sites and NetOGlyc services for predicting O-linked glycosylation sites).
  • an aglycosyl anti-CD8 antibody is incapable of depleting CD8- bearing cells in mice (Isaacs, 1992 J. Immunol. 148: 3062) and an aglycosyl anti-CD3 antibody does not induce cytokine release syndrome in mice or humans (Boyd, 1995 supra; Friend, 1999 Transplantation 68: 1632).
  • the anti-human S 1P4 antibodies of the present invention may be modified or altered to elicit reduced effector function(s) (compared to a second S lP4-specific antibody) while optionally retaining the other valuable attributes of the Fc portion.
  • the present invention relates to aglycosyl anti- human S1P4 antibodies with decreased effector function, which are characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of the antibody.
  • a modification of the conserved N-linked site in the CH2 domains of the Fc dimer can lead to aglycosyl anti-SlP4 antibodies. Examples of such modifications include mutation of the conserved N-linked site in the CH2 domains of the Fc dimer, removal of glycans attached to the N-linked site in the CH2 domains, and prevention of glycosylation.
  • an aglycosyl anti-SlP4 antibody may be created by changing the canonical N- linked Asn site in the heavy chain CH2 domain to a Gin residue (see, for example, WO 05/03175 and US 2006-0193856).
  • the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at the site.
  • the aglycosyl anti-SlP4 antibodies are prepared by mutation of the heavy chain glycosylation site, i.e., mutation of N298Q ( 297 using Kabat EU numbering) and expressed in an appropriate host cell.
  • this mutation may be accomplished by following the manufacturer's recommended protocol for unique site mutagenesis kit from Amersham-Pharmacia Biotech® (Piscataway, NJ, USA).
  • the mutated antibody can be stably expressed in a host cell (e. g. NSO or CHO cell) and then purified.
  • a host cell e. g. NSO or CHO cell
  • purification can be carried out using Protein A and gel filtration chromatography. It will be apparent to those of skill in the art that additional methods of expression and purification may also be used.
  • the aglycosyl anti-human S1P4 antibodies have decreased effector function, wherein the modification at the conserved N- linked site in the CH2 domains of the Fc portion of said antibody or antibody derivative comprises the removal of the CH2 domain glycans, i.e., deglycosylation.
  • These aglycosyl anti-S lP4 antibodies may be generated by conventional methods and then deglycosylated enzymatically. Methods for enzymatic deglycosylation of antibodies are well known to those of skill in the art (Williams, 1973; Winkelhake & Nicolson, 1976 J. Biol Chem., 251 : 1074- 80).
  • deglycosylation may be achieved by growing host cells which produce the antibodies in culture medium comprising a glycosylation inhibitor such as tunicamycin (Nose & Wigzell, 1983). That is, the modification is the reduction or prevention of glycosylation at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody.
  • a glycosylation inhibitor such as tunicamycin (Nose & Wigzell, 1983).
  • recombinant human S1P4 polypeptides may be used as an antigen to generate an anti-SlP4 antibody or antibody derivatives, which may then be deglycosylated.
  • agyclosyl anti-human S1P4 antibodies or anti-human S1P4 antibodies with reduced glycosylation may be produced by the method described in Taylor et al. (WO 05/18572 and US 2007-0048300).
  • an anti-S lP4 aglycosyl antibody may be produced by altering a first amino acid residue (e.g., by substitution, insertion, deletion, or by chemical modification), wherein the altered first amino acid residue inhibits the glycosylation of a second residue by either steric hindrance or charge or both.
  • the first amino acid residue is modified by amino acid substitution.
  • the amino acid substitution is selected from the group consisting of Gly, Ala, Val, Leu, He, Phe, Asn, Gin, Trp, Pro, Ser, Thr, Tyr, Cys, Met, Asp, Glu, Lys, Arg, and His.
  • the amino acid substitution is a non- traditional amino acid residue.
  • the second amino acid residue may be near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS.
  • the first amino acid residue is amino acid 299 and the second amino acid residue is amino acid 297, according to the Kabat numbering.
  • the first amino acid substitution may be T299A, T299N, T299G, T299Y, T299C, T299H, T299E, T299D, T299K, T299R, T299G, T299I, T299L, T299M, T299F, T299P, T299W, and T299V, according to the Kabat numbering.
  • the amino acid substitution is T299C.
  • Effector function may also be reduced by modifying an antibody of the present invention such that the antibody contains a blocking moiety.
  • exemplary blocking moieties include moieties of sufficient steric bulk and/or charge such that reduced glycosylation occurs, for example, by blocking the ability of a glycosidase to glycosylate the polypeptide.
  • the blocking moiety may additionally or alternatively reduce effector function, for example, by inhibiting the ability of the Fc region to bind a receptor or complement protein.
  • the present invention relates to an SlP4-binding protein, e.g., an anti-SlP4 antibody, comprising a variant Fc region, the variant Fc region comprising a first amino acid residue and an N-glycosylation site, the first amino acid residue modified with side chain chemistry to achieve increased steric bulk or increased electrostatic charge compared to the unmodified first amino acid residue, thereby reducing the level of or otherwise altering glycosylation at the N-glycosylation site.
  • the variant Fc region confers reduced effector function compared to a control, non-variant Fc region.
  • the side chain with increased steric bulk is a side chain of an amino acid residue selected from the group consisting of Phe, Trp, His, Glu, Gin, Arg, Lys, Met and Tyr.
  • the side chain chemistry with increased electrostatic charge is a side chain of an amino acid residue selected from the group consisting of Asp, Glu, Lys, Arg, and His. Accordingly, in one embodiment, glycosylation and Fc binding can be modulated by substituting T299 with a charged side chain chemistry such as D, E, K, or R. The resulting antibody will have reduced glycosylation as well as reduced Fc binding affinity to an Fc receptor due to unfavorable electrostatic interactions.
  • a T299C variant antibody which is both aglycosylated and capable of forming a cysteine adduct, may exhibit less effector function (e.g., FcyRI binding) compared to its aglycosylated antibody counterpart (see, e.g., WO 05/18572). Accordingly, alteration of a first amino acid proximal to a glycosylation motif can inhibit the glycosylation of the antibody at a second amino acid residue; when the first amino acid is a cysteine residue, the antibody may exhibit even further reduced effector function.
  • inhibition of glycosylation of an antibody of the IgG4 subtype may have a more profound effect on FcyRI binding compared to the effects of aglycosylation in the other subtypes.
  • glycosylation structures on a glycoprotein will vary depending upon the expression host and culturing conditions (Raju, TS. BioProcess International April 2003. 44-53). Such differences can lead to changes in both effector function and pharmacokinetics (Israel et al. Immunology. 1996; 89(4):573-578; Newkirk et al. P. Clin. Exp. 1996;
  • galactosylation can vary with cell culture conditions, which may render some immunoglobulin compositions immunogenic depending on their specific galactose pattern (Patel et al, 1992. Biochem J. 285: 839-845).
  • the oligosaccharide structures of glycoproteins produced by non-human mammalian cells tend to be more closely related to those of human glycoproteins.
  • protein expression host systems may be engineered or selected to express a predominant Ig glycoform or alternatively may naturally produce glycoproteins having predominant glycan structures.
  • Examples of engineered protein expression host systems producing a glycoprotein having a predominant glycoform include gene knockouts/mutations (Shields et al, 2002, JBC, 277: 26733-26740); genetic engineering in (Umana et al, 1999, Nature Biotech., 17: 176-180) or a combination of both.
  • certain cells naturally express a predominant glycoform— for example, chickens, humans and cows (Raju et al., 2000, Glycobiology , 10: 477-486).
  • an anti-S lP4 antibody or antibody composition having altered glycosylation e.g., predominantly one specific glycan structure
  • Protein expression host systems that may be used to produce anti-SlP4 antibodies of the present invention include animal, plant, insect, bacterial cells and the like.
  • US 2007-0065909, 2007-0020725, and 2005-0170464 describe producing aglycosylated immunoglobulin molecules in bacterial cells.
  • Wright and Morrison produced antibodies in a CHO cell line deficient in glycosylation (1994 J Exp Med 180: 1087-1096) and showed that antibodies produced in this cell line were incapable of complement-mediated cytolysis.
  • Other examples of expression host systems found in the art for production of glycoproteins include: CHO cells: Raju WO 99/22764 and Presta WO 03/35835; hybridoma cells: Trebak et al, 1999, J.
  • the aglycosyl anti-human S1P4 antibodies with reduced effector function may be antibodies that comprise modifications or that may be conjugated to comprise a functional moiety.
  • Such moieties include a blocking moiety (e.g., a PEG moiety, cysteine adducts, etc.), a detectable moiety (e.g., fluorescent moieties, radioisotopic moieties, radiopaque moieties, etc., including diagnostic moieties), a therapeutic moiety (e.g., anti-inflammatory agents, immunomodulatory agents, anti-infective agents, anti-neurodegenerative agents, etc.), and/or a binding moiety or bait (e.g., that allows the antibody to be pre-targeted to a cell and then to bind a second molecule, composed of the complementary binding moiety or prey and a detectable moiety or therapeutic moiety, as described above).
  • a blocking moiety e.g., a PEG moiety, cysteine adducts
  • the anti-human S1P4 antibodies or antigen-binding fragments thereof described herein can be used in the diagnosis and treatment, including prevention, of demyelinating diseases.
  • the antibodies or antigen-binding fragments thereof can be conjugated to an agent (e.g., siRNA, miRNA, anti-miR, small molecule, or other small molecule chemical moiety) that is useful to treat or prevent the disease being treated or prevented with the anti-SlP4 antibodies or antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof described herein can be used to promote oligodendrocyte differentiation.
  • the antibodies or antigen-binding fragments thereof described herein can be used to promote myelination and/or inhibit demyelination. In other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to enhance myelin repair. In further embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to promote motor neuron survival. In yet other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to promote axon integrity.
  • the antibodies or antigen-binding fragments thereof of this disclosure can be used to diagnose, treat, or prevent a demyelinating disease or disorder of the central nervous system (CNS) or the peripheral nervous system (PNS).
  • Demyelinating diseases or disorders of the CNS include multiple sclerosis, Devic's disease (also known as Neuromyelitis optica), acute disseminated encephalomyelitis (ADEM), acute hemorrhagic leukoencephalitis (AHL), inflammatory demyelinating diseases (e.g., cerebellitis, transverse myelitis, brain stem encephalitis, optic neuritis), CNS sarcoidosis, CNS neuropathies (e.g., those produced by Vitamin B 12 deficiency), viral encephalitis, paraneoplastic encephalitis, central pontine myelinolysis, myelopathies (e.g., Tabes dorsalis); leukoencephalopathies (e.g., progressive multifocal le
  • the methods comprise preventing and/or treating the disease, preventing the onset of the disease, slowing or reversing the progress of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease, preventing reducing, or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.
  • the antibodies or antigen-binding fragments thereof of this disclosure can be used to diagnose, treat, or prevent multiple sclerosis. In another embodiment, the antibodies or antigen-binding fragments thereof of this disclosure can be used to diagnose, treat, or prevent amyotrophic lateral sclerosis.
  • Multiple sclerosis is widespread, affecting approximately 2.5 million people worldwide. Multiple sclerosis is associated with the destruction of myelin,
  • oligodendrocytes, or axons localized to chronic lesions The demyelination observed in multiple sclerosis may not always be permanent and remyelination has been documented in early stages of the disease. Relapsing remitting multiple sclerosis is the most common form of the disease. The title can be misleading. During this form of the disease, patients tend to experience an attack or series of attacks (exacerbations) followed by complete or partial remission. Patients often assume that the remission stage of the disease will mean 100% recovery. While this can be true, particularly during the early stages of the disease, the remission is often only partial, particularly as the disease progresses, leaving the patient with residual, usually permanent, symptoms. Primary progressive multiple sclerosis is most commonly found in men.
  • Secondary progressive multiple sclerosis begins with relapsing remitting multiple sclerosis.
  • the relapsing remitting (rrms) stage of the disease may persist for many years before the onset of secondary progressive multiple sclerosis.
  • Secondary progressive multiple sclerosis is a second-stage, chronic, progressive form of the disease where, unlike the relapsing remitting stage, there are no real periods of remission, only breaks in attack duration with no real recovery from symptoms although there may be minor relief from some.
  • Progressive relapsing multiple sclerosis is a rarer form of multiple sclerosis where the disease takes a progressive form from the outset with acute attacks throughout and no relief from
  • progressive relapsing multiple sclerosis does not "plateau.”
  • the antibodies or antigen-binding fragments thereof described herein can be used to treat relapsing remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and progressive relapsing multiple sclerosis.
  • animal models of multiple sclerosis include the purely autoimmune experimental autoimmune encephalomyelitis (see, e.g., Simmons et al, Trends Immunol, 34(8):410-422 (2013); and Furlan et al, Methods Mol.
  • An anti-SlP4 antibody or antigen-binding fragment thereof described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disorder described herein (e.g., multiple sclerosis or amyotrophic lateral sclerosis).
  • the anti- S1P4 antibody or antigen-binding fragment thereof may be conjugated (e.g., to a siRNA, miRNA, anti-miR, small molecule, or other chemical moiety).
  • a pharmaceutical composition includes a pharmaceutically acceptable carrier.
  • composition includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19).
  • compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the preferred form can depend on the intended mode of administration and therapeutic application.
  • compositions for the agents described herein are in the form of injectable or infusible solutions.
  • an anti-SlP4 antibody described herein is formulated with excipient materials, such as sodium citrate, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, Tween-80, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8°C.
  • the pH of the composition is between about 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5).
  • the pharmaceutical compositions can also include agents that reduce aggregation of the S1P4 antibody or antigen-binding fragment thereof when formulated.
  • aggregation reducing agents include one or more amino acids selected from the group consisting of methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. These amino acids may be added to the formulation to a concentration of about 0.5 mM to about 145 mM (e.g., 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM).
  • the pharmaceutical compositions can also include a sugar (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and/or a tonicity modifier (e.g., sodium chloride, mannitol, or sorbitol) and/or a surfactant (e.g., polysorbate-20 or polysorbate-80).
  • a sugar e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol
  • a tonicity modifier e.g., sodium chloride, mannitol, or sorbitol
  • a surfactant e.g., polysorbate-20 or polysorbate-80.
  • compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection).
  • a parenteral mode e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection.
  • the anti-SlP4 antibody or antigen-binding fragment thereof compositions are administered by inhalation.
  • the anti-SlP4 antibody or antigen-binding fragment thereof are administered by inhalation.
  • compositions are administered subcutaneously.
  • the anti-SlP4 antibody or antigen-binding fragment thereof compositions are administered intravenously.
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration.
  • Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the anti-SlP4 antibody or antigen-binding fragment thereof may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems.
  • a controlled release formulation including implants, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York (1978).
  • the pharmaceutical formulation comprises an anti-SlP4 antibody or antigen-binding fragment thereof at a concentration of about 0.5 mg/mL to 500 mg/mL (e.g., 0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/ mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 350 mg/mL, 400 mg/mL, 450 mg/mL, 500 mg/mL), formulated with a pharmaceutically acceptable carrier.
  • the anti-S lP4 antibody or antigen-binding fragment thereof is formulated in sterile distilled water or phosphate buffered saline.
  • the pH of the pharmaceutical formulation may be between 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 6.3, 6.4 6.5, 6.6 6.7, 6.8, 6.9 7.0, 7.1, 7.3, 7.4, 7.5).
  • the anti-human S 1P4 antibody or antigen-binding fragment thereof can be administered to a subject, e.g., a subject in need thereof, for example, a human subject, by a variety of methods.
  • the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), inhalation, or intramuscular injection. It is also possible to use intra-articular delivery. Other modes of parenteral administration can also be used.
  • administration can be oral.
  • the route and/or mode of administration of the antibody or antigen-binding fragment thereof can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using tomographic imaging, e.g., to visualize a tumor.
  • the antibody or antigen-binding fragment thereof can be administered as a fixed dose, or in a mg kg dose.
  • the dose can also be chosen to reduce or avoid production of antibodies against the anti-SlP4 antibody.
  • Dosage regimens are adjusted to provide the desired response, e.g., a therapeutic response or a combinatorial therapeutic effect.
  • doses of the anti-human S1P4 antibody or antigen binding fragment thereof (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities.
  • doses in the range of 0.1-100 mg/kg, 0.5-100 mg/kg, 1 mg/kg -100 mg/kg, 0.5-20 mg/kg, 0.1-10 mg/kg, or 1-10 mg/kg can be administered.
  • a subject in need of treatment with an anti-human S1P4 antibody or antigen binding fragment thereof is administered the antibody at a dose of 1 mg/kg to 30 mg/kg.
  • a subject in need of treatment with an anti-human S1P4 antibody or antigen-binding fragment thereof is administered the antibody at a dose of 1 mg/kg, 2 mg/kg, 4 mg/kg, 5 mg/kg, 7 mg/kg 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 28 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, or 50 mg/kg.
  • the antibodies or antigen-binding fragments thereof are administered intravenously at a dose of 4 mg/kg to 30 mg/kg.
  • a composition may comprise about 1 mg/mL to 100 mg/ml or about 10 mg/mL to 100 mg/ml or about 50 to 250 mg/mL or about 100 to 150 mg/ml or about 100 to 250 mg/ml of anti-SlP4 antibody or an antigen-binding fragment thereof.
  • the anti-S lP4 antibody or antigen-binding fragment thereof in a composition is predominantly in monomeric form, e.g., at least about 90%, 92%, 94%, 96%, 98%, 98.5% or 99% in monomeric form.
  • Certain anti -human S1P4 antibody or antigen-binding fragment thereof compositions may comprise less than about 5, 4, 3, 2, 1, 0.5, 0.3 or 0.1% aggregates, as detected, e.g., by UV at A280 nm. Certain anti-human S 1P4 antibody or antigen-binding fragment thereof compositions comprise less than about 5, 4, 3, 2, 1, 0.5, 0.3, 0.2 or 0.1% fragments, as detected, e.g., by UV at A280 nm.
  • Dosage unit form or "fixed dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of anti-SlP4 antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, the antibody may be administered via continuous infusion.
  • An anti-human S 1P4 antibody or antigen-binding fragment thereof dose can be administered, e.g., at a periodic interval over a period of time (a course of treatment) sufficient to encompass at least 2 doses, 3 doses, 5 doses, 10 doses, or more, e.g., once or twice daily, or about one to four times per week, or preferably weekly, biweekly (every two weeks), every three weeks, monthly, e.g., for between about 1 to 12 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • Factors that may influence the dosage and timing required to effectively treat a subject include, e.g., the severity of the disease or disorder, formulation, route of delivery, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments.
  • the antibody can be administered before the full onset of the disorder, e.g., as a preventative measure.
  • the duration of such preventative treatment can be a single dosage of the antibody or the treatment may continue (e.g., multiple dosages).
  • a subject at risk for the disorder or who has a predisposition for the disorder may be treated with the antibody for days, weeks, months, or even years so as to prevent the disorder from occurring or fulminating.
  • a pharmaceutical composition may include a "therapeutically effective amount" of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used.
  • a therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.
  • the anti-human S 1P4 antibody or antigen-binding fragment thereof is administered subcutaneous ly at a concentration of about 1 mg/mL to about 500 mg/mL (e.g., 1 mg/mL, 2 mg/mL, 3 mg/mL 3 4 mg/mL t 5 mg/mL , 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/niL, 65 mg/niL, 70 mg/niL, 75 mg/niL, 80 mg/niL, 85 mg/niL, 90 mg/niL, 95 mg/niL, 100 mg/niL, 125 mg/niL, 150 mg/niL, 175 mg/niL, 200 mg/niL, 225 mg/niL, 250 mg/niL, 275 mg
  • the anti-SlP4 antibody or antigen-binding fragment thereof is administered subcutaneously at a concentration of 50 mg/mL. In another embodiment, the anti-human S1P4 antibody or antigen-binding fragment thereof is administered intravenously at a concentration of about 1 mg/mL to about 500 mg/mL. In a particular embodiment, the anti- S1P4 antibody or antigen-binding fragment thereof is administered intravenously at a concentration of 50 mg/mL.
  • the anti-human S 1P4 antibody or antigen-binding fragment thereof can be administered to a patient in need thereof (e.g., a patient having a demyelinating disease) in combination with another antagonist (e.g., antibody, polypeptide antagonist (e.g., dominant- negative), siRNA, and/or small molecule antagonist) of human S1P4.
  • another antagonist e.g., antibody, polypeptide antagonist (e.g., dominant- negative), siRNA, and/or small molecule antagonist
  • a patient in need thereof e.g., a patient having a demyelinating disease
  • another antagonist e.g., antibody, polypeptide antagonist (e.g., dominant- negative), siRNA, and/or small molecule antagonist) of human S1P4.
  • another antagonist e.g., antibody, polypeptide antagonist (e.g., dominant- negative), siRNA, and/or small molecule antagonist) of human S1P4.
  • the anti-human S1P4 antibody or antigen-binding fragment thereof is conjugated to a miRNA, siRNA, anti-miR, small molecule, or other chemical moiety.
  • the anti-human S 1P4 antibody or antigen-binding fragment thereof can be administered to a patient in need thereof (e.g., a patient having a demyelinating disease, e.g., multiple sclerosis) in combination with any one or more of: corticosteroids (e.g., oral prednisone and intravenous methylprednisolone (Solu-Medrol)); beta interferons (e.g., interferon ⁇ -la (Avonex), interferon ⁇ -lb (Betaseron), interferon ⁇ -lb (Extavia), and interferon ⁇ -la (Rebif)); glatiramer acetate (Copaxone); fingolimod (Gilenya); natalizumab (Tysabri); dimethyl fumarate (Tecfidera); mitoxantrone ( ovantrone); triflunomide
  • corticosteroids e.g., oral pre
  • dalfampridine Ampyra
  • muscle relaxants e.g., baclofen (Lioresal) and tizanidine (Zanaflex)
  • medications to reduce fatigue e.g., amantadine
  • the anti-human S1P4 antibody or antigen- binding fragment thereof can be administered to the patient in need of the treatment before, substantially at the same time as, or after the other treatment(s).
  • compositions that include the anti-SlP4 antibody or antigen-binding fragment thereof can be administered with a medical device.
  • the device can be designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed from medical facilities and other medical equipment.
  • the device can include, e.g., one or more housings for storing pharmaceutical preparations that include anti-SlP4 antibody or antigen-binding fragment thereof, and can be configured to deliver one or more unit doses of the antibody.
  • the device can be further configured to administer a second agent, e.g., a chemo therapeutic agent, either as a single pharmaceutical composition that also includes the anti-S lP4 antibody or antigen-binding fragment thereof or as two separate pharmaceutical compositions.
  • the pharmaceutical composition may be administered with a syringe.
  • the pharmaceutical composition can also be administered with a needleless hypodermic injection device, such as the devices disclosed in US 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556.
  • implants and modules examples include: US 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486, 194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439, 196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475, 196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.
  • kits An anti-human S1P4 antibody or antigen-binding fragment thereof can be provided in a kit.
  • the kit includes (a) a container that contains a composition that includes anti-human S1P4 antibody or antigen-binding fragment thereof, and optionally (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit.
  • the kit also includes at least one second agent for treating a disorder described herein (e.g., an antagonist (e.g., antibody, polypeptide antagonist, siRNA, and/or small molecule antagonist) of human S 1P4.
  • the at least one second agent is selected from the group consisting of corticosteroids (e.g., oral prednisone and intravenous methylprednisolone (Solu-Medrol)); beta interferons (e.g., interferon ⁇ -la (Avonex), interferon ⁇ -lb (Betaseron), interferon ⁇ -lb (Extavia), and interferon ⁇ -la (Rebif)); glatiramer acetate (Copaxone); fingolimod (Gilenya); natalizumab (Tysabri);
  • corticosteroids e.g., oral prednisone and intravenous methylprednisolone (Solu-Medrol)
  • dalfampridine Ampyra
  • muscle relaxants e.g., baclofen (Lioresal) and tizanidine
  • the kit includes a first container that contains a composition that includes the anti-human S1P4 antibody or an antigen-binding fragment thereof, and a second container that includes the second agent.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods of administering the anti -human S1P4 antibody or antigen-binding fragment thereof, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has had or who is at risk for an immunological disorder described herein.
  • the information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material, e.g., on the internet.
  • composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative.
  • the antibody can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution.
  • the antibody or antigen binding fragment thereof in the liquid solution is at a concentration of about 25 mg/mL to about 250 mg/mL (e.g., 40 mg/mL, 50 mg/mL, 60 mg/mL, 75 mg/mL, 85 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 200 mg/mL).
  • the antibody or antigen binding fragment is provided as a lyophilized product, the antibody or antigen binding fragment is at about 75 mg/vial to about 200 mg/vial (e.g., 100 mg/vial, 108.5 mg/vial, 125 mg/ vial, 150 mg/vial).
  • the lyophilized powder is generally reconstituted by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer (e.g., phosphate buffered saline), can optionally be provided in the kit.
  • the lyophilized product is at 108.5 mg/vial and reconstituted to a liquid solution at a concentration of 75 mg/mL.
  • the kit can include one or more containers for the composition or compositions containing the agents.
  • the kit contains separate containers, dividers or compartments for the composition and informational material.
  • the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet.
  • the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents.
  • the containers can include a combination unit dosage, e.g., a unit that includes both the anti-human S 1P4 antibody or antigen-binding fragment thereof and the second agent, e.g., in a desired ratio.
  • the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose.
  • the containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • the kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device.
  • a device suitable for administration of the composition e.g., a syringe or other suitable delivery device.
  • the device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.
  • Anti-human 1P4 antibodies or antigen-binding fragments thereof can be used in a diagnostic method for detecting the presence of S1P4 in vivo (e.g., in vivo imaging in a subject).
  • anti-SlP4 antibodies can be administered to a subject to detect S1P4 within the subject.
  • the antibody can be labeled, e.g., with an MRI detectable label or a radiolabel.
  • the subject can be evaluated using a means for detecting the detectable label.
  • the subject can be scanned to evaluate localization of the antibody within the subject.
  • the subject is imaged, e.g., by NMR or other tomographic means.
  • labels useful for diagnostic imaging include radiolabels such as 131 I, u l In, 123 1, 99m Tc, 32 P, 33 P, 125 1, 3 H, 14 C, and 188 Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase.
  • Short-range radiation emitters such as isotopes detectable by short-range detector probes, can also be employed.
  • the protein ligand can be labeled with such reagents using known techniques. For example, see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for techniques relating to the radiolabeling of antibodies and Colcher et al. (1986) Meth.
  • the subject can be "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al, "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al, (eds.), pp 65-85 (Academic Press 1985).
  • a positron emission transaxial tomography scanner such as designated Pet VI located at Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., U C, 18 F, 15 0, and 13 N).
  • Magnetic Resonance Imaging uses NMR to visualize internal features of living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit.
  • Some MRI techniques are summarized in EPO 502 814 A. Generally, the differences related to relaxation time constants Tl and T2 of water protons in different environments are used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.
  • contrast agents include a number of magnetic agents, paramagnetic agents (which primarily alter Tl) and ferromagnetic or superparamagnetic agents (which primarily alter T2 response).
  • Chelates e.g., EDTA, DTPA and NTA chelates
  • Other agents can be in the form of particles, e.g., less than 10 ⁇ to about 10 nm in diameter).
  • Particles can have ferromagnetic, anti-ferromagnetic or superparamagnetic properties.
  • Particles can include, e.g., magnetite (Fe30 4 ), y-Fe203, ferrites, and other magnetic mineral compounds of transition elements.
  • Magnetic particles may include one or more magnetic crystals with and without nonmagnetic material.
  • the nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like).
  • the anti-SlP4 antibodies or antigen-binding fragments thereof can also be labeled with an indicating group containing the NMR-active 19 F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the 19 F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American, 246:78-88 to locate and image S 1P4 distribution.
  • the disclosure provides a method for detecting the presence of S1P4 in a sample in vitro (e.g., a biological sample, such as tissue, biopsy).
  • This method can be used to assay the expression level of S1P4.
  • the method includes: (i) contacting the sample or a control sample with the anti-SlP4 antibody or an antigen-binding fragment thereof; and (ii) evaluating the sample for the presence of S1P4, e.g., by detecting formation of a complex between the anti-SlP4 antibody and S1P4.
  • the antibody can be immobilized, e.g., on a support, and retention of the antigen on the support is detected, and/or vice versa.
  • the antibody used may be labeled e.g., with a fluorophore.
  • a control sample can be included.
  • a statistically significant change in the formation of the complex in the sample relative to the control sample can be indicative of the presence of S1P4 in the sample.
  • an anti-SlP4 antibody can be used in applications that include fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and cell sorting (e.g., fluorescence activated cell sorting).
  • the anti-S lP4 antibody is a 1F1 1 IgG2 antibody or an antigen-binding fragment thereof.
  • Assaying S 1P4 expression levels in a biological sample can occur using any art- known method.
  • Preferred for assaying S1P4 expression levels in a biological sample are immunological techniques.
  • S 1P4 expression in tissues can be studied with classical immunohistological methods.
  • the specific recognition is provided by a primary ligand, e.g., an antibody (polyclonal or monoclonal), that binds to S 1P4.
  • This primary ligand can be labeled, e.g., with a fluorescent, chemiluminescent, phosphorescent, enzymatic or radioisotopic label.
  • these methods of the invention can use a secondary detection system in which a second ligand that recognizes and binds to the S 1P4- binding ligand, e.g., a so-called "secondary" antibody which recognizes and binds to a first SlP4-binding antibody, is detectably labeled as described above.
  • a second ligand that recognizes and binds to the S 1P4- binding ligand e.g., a so-called "secondary” antibody which recognizes and binds to a first SlP4-binding antibody
  • tissues and cell samples can also be extracted, e.g., with urea and neutral detergent, for the liberation of S1P4 protein for Western-blot or dot/slot assay (Jalkanen, M., et al, J. Cell. Biol, 101 :976-985 (1985); Jalkanen, M., et al, J. Cell. Biol, 105:3087-3096 (1987)) for direct quantitation, relative to a standard tissue or cell sample known to have lower levels of expression of S1P4.
  • Western-blot or dot/slot assay Jalkanen, M., et al, J. Cell. Biol, 101 :976-985 (1985); Jalkanen, M., et al, J. Cell. Biol, 105:3087-3096 (1987)
  • This disclosure also provides methods of screening for antagonists of S1P4.
  • the method comprises contacting neuronal cells (e.g., neurons, oligodendrocyte precursor cells), or a neuronal cell line expressing S 1P4 (e.g., SK-N-MC, SK-N-SH, neuroblastoma cell lines), or a non-neuronal cell line expressing S1P4 (e.g., Jurkat, breast cancer cell lines), with a test agent (e.g., an antibody, small molecule inhibitor, a polypeptide).
  • a test agent e.g., an antibody, small molecule inhibitor, a polypeptide
  • test agent increases the expression of myelin basic protein (MBP) and/or myelin oligodendrocyte glycoprotein (MOG) compared with a negative control (e.g., an antibody known to not increase MBP or MOG levels), and/or if the test agent increases the expression of myelin basic protein (MBP) and/or myelin oligodendrocyte glycoprotein (MOG) to the same extent, or about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a positive control (e.g., 1F1 1 IgG2), the agent is determined as an antagonist of S 1P4.
  • a positive control e.g., 1F1 1 IgG2
  • expression of MBP and MOG can be determined by any method known in the art, e.g., Western blot analysis.
  • Such antagonists are useful in promoting oligodendrocyte proliferation, promoting myelination, inhibiting demyelination, promoting motor neuron survival, promoting axon integrity, treating demyelinating disorders such as multiple sclerosis, and treating amyotrophic lateral sclerosis
  • Example 1 S1P4 mRNA Expression in Neuronal Cells mRNAs were extracted from the primary cells or cell lines being studied using a Stratagene Kit. mRNAs from human tissues were ordered from Clontech. Taqman RT-PCR was used to quantify S1P4 mRNA levels, ⁇ -actin was used as an internal control. All the primer sets were ordered from Applied Biosystem.
  • S1P4 expression in rat neuronal cells, human neuronal and tumor cell lines, and human tissues was evaluated by Taqman RT-PCR quantification methods.
  • S1P4 mRNA levels are highest in the cortical neurons, intermediate in OPC and motor neurons, and lowest in astrocyte, DRG neurons and Schwann cells (SWC) ( Figures 1 and 2).
  • S1P4 mRNA levels are highest in neurons and oligodendrocytes (OPC), intermediate in neuroblastoma cells (SK-N-MC and SK-N-SH) and Jurkat T cells (an immortalized line of T lymphocyte cells), and lowest in neuronal glioblastoma U 138 MG and U97 MG cell lines; S1P4 mRNA is not detectable in human astrocytes ( Figure 3).
  • S1P4 mRNA levels are highest in human thymus and lung, intermediate in liver and muscle, and lowest in brain (Figure 4).
  • oligodendrocytes from female Sprague Dawley post-natal day 2 (P2) rats were grown in culture.
  • the forebrain was dissected and placed in Hank's buffered salt solution (HBSS) (Life Technologies). The tissue was cut into 1 mm fragments and incubated at 37°C for 15 min in 0.01% trypsin and 10 ⁇ g/ml DNase.
  • Dissociated cells were plated on poly-L-lysine-coated T75 tissue culture flasks and grown at 37°C for 10 days in Dulbecco's Modified Eagle Medium (DMEM) with 20% fetal calf serum (Life
  • DMEM Dulbecco's Modified Eagle Medium
  • Oligodendrocyte precursors (A2B5 + ) were collected by shaking the flask overnight at 200 rpm at 37°C, resulting in a 95% pure population. Cultures were maintained in high glucose DMEM with 10 ng/ml fibroblast growth factor/platelet-derived growth factor (FGF/PDGF) (Peprotech) for 1 week.
  • FGF/PDGF fibroblast growth factor/platelet-derived growth factor
  • A2B5 + progenitor cells were plated into 4-well slide chambers or 24-well culture plate in FGF/PDGF free growth medium supplemented with N2, and were immediately treated for 72 hr with siRNA, lentivirus containing full length, or S1P4 dominant negative (DN).
  • MBP + myelin basic protein positive
  • siRNA for rat S1P4 was ordered from Thermo Scientific as a pool of 4 Target Sequences: GGGACAGGGCCUUACGGUU (SEQ ID NO: 60), CCGGGUUCCUUCCACGAAA (SEQ ID NO: 61), UGACAGUACAGCUGCGAAC (SEQ ID NO: 62), and CCUGAUGUGUUGUAAGAAC (SEQ ID NO: 63).
  • Cultures were subjected to immunocytochemical (ICC) analysis for MBP expression, MBP/MOG Western blotting and MBP MSD assay.
  • ICC immunocytochemical
  • S1P4 The effect of blocking S1P4 was studied in vitro by transfecting primary rat forebrain A2B5 + OPCs with an S1P4 RNAi or an S1P4 DN construct. Seventy-two hours after transfection, cultures were stained for MBP expression by ICC or lysed for Western blotting ( Figure 5 and 6) for monitoring differentiation. OPCs transfected with S 1P4 RNAi or S1P4 DN resulted in more highly differentiated, mature oligodendrocytes by ICC as evident by the increased length of their cell processes and the presence of abundant myelin sheath structures compared to controls ( Figure 5 and 6).
  • spinal cords from S1P4 knockout mice contained more myelinated axon fibers at early stages (P2 and P6) compared with wild- type mice, while no difference were observed at later stages (PI 5 and P60).
  • S1P4 DN was used in a dorsal root ganglia (DRG)/OPC co-culture assay to validate key aspects of S1P4 biology on myelination in vitro.
  • Co-culture is a unique system for studying the myelination of axons by oligodendrocytes in an in vitro setting.
  • rat primary El 6 DRG cells and P2 A2B5 + oligodendrocyte progenitor cells were co-cultured for 10 days in the presence and absence of S 1P4 DN.
  • Myelination was determined by Western blotting to quantify MBP and MOG levels.
  • S1P4 DN expression in OPCs resulted in robust myelination as evident by the increase in MBP and MOG protein levels (Figure 10).
  • S1P4 DN expression in DRG cells did not promote myelination, indicating that S1P4 DN promotes myelination by blocking the effect of S1P4 on OPCs, but not DRG.
  • RhoA signaling pathway has been known to be a negative regulator of OPC differentiation. To validate that blocking S1P4 by S1P4 DN is able to block RhoA signaling pathway which leads to OPC differentiation, a RhoA activation study was performed.
  • CHO-K1 cells Approximately 5 million CHO-K1 cells were transfected with S1P4 FL, S1P4 DN, or GFP (control). Cells were cultured for 48 hours and then treated with SIP ( ⁇ ) in serum- free media containing 0.1% fatty acid free Bovine serum albumin (BSA) for 5 or 20 min. Cells were then lysed and Western blotting was performed to detect activated RhoA levels (Figure 11). Compared to control GFP expression cells, S 1P4 FL expression cells showed an increase in activated RhoA levels. In contrast, S1P4 DN expression cells showed a decrease in activated RhoA levels.
  • SIP serum-free media containing 0.1% fatty acid free Bovine serum albumin
  • a phage display library was constructed containing antibodies with mutations in the CDR-H2 and/or CDR-L3 of a parental antibody clone termed 1F7.
  • An anti-SlP4 antibody, 1F1 1 was selected after 7 rounds of bio-panning of the phage display library against a N- terminal peptide (RLIVLHYNHSGRLAGRGGPEDGGLGALR (SEQ ID NO:4)) of human S 1P4.
  • the light chain and heavy chains of the selected antibody clone were converted to full length human IgG format, expressed in 293 cells (transient system), purified by protein A chromatography, and tested for affinities by ELISA against the N-terminal peptide and by Western against human S1P4 protein transiently produced in 293 cells.
  • the best performer was produced at a large scale and further tested in an OPC differentiation assay (see below).
  • oligodendrocytes from Female Long Evans post-natal day 2 (P2) rats were grown in culture. The forebrain was dissected and placed in Hank's buffered salt solution (HBSS) (Invitrogen, Grand Island, NY). The tissue was cut into 1 mm fragments and incubated at 37°C for 15 min in 0.01% trypsin and 10 ⁇ g/ml DNase.
  • HBSS Hank's buffered salt solution
  • Dissociated cells were plated on poly-L-lysine-coated T75 tissue culture flasks and grown at 37°C for 10 days in DME medium with 20% fetal calf serum (Invitrogen). Oligodendrocyte precursors (A2B5 + ) were collected by shaking the flask overnight at 200 rpm at 37°C, resulting in a 95% pure population. Cultures were maintained in high glucose DME medium with FGF/PDGF (Peprotech) (10 ng/ml) for 1 week. For assessing the ability of a testing reagent to promote the differentiation of rat A2B5 + progenitor cells into mature myelin basic protein positive (MBP + ) myelinating oligodendrocytes.
  • MBP + myelinating oligodendrocytes.
  • A2B5 + cells were plated into 4-well slide chambers or 24 well culture plate in FGF/PDGF free growth medium supplemented with T3/CNTF, and immediately treating cultures for 72 hours with 10 ⁇ g/ml of anti-S lP4 or a control antibody (5c8, the anti-CD40L antibody). Oligodendrocyte differentiation was determined by MBP/MOG Western. The anti-SlP4 antibody (1F1 1 IgG2) promoted oligodendrocyte differentiation, as shown by MAG and MBP expression on the Western blots ( Figure 12).

Abstract

La présente invention concerne des anticorps ainsi que des fragments associés d'anticorps qui se lient spécifiquement au récepteur de la sphingosine-1-phosphate 4 (S1P4). L'invention concerne également des méthodes d'utilisation des anticorps et des fragments d'anticorps anti-S1P4 en vue de traiter ou de prévenir des maladies neurologiques telles que la sclérose en plaques ou la sclérose latérale amyotrophique.
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