HK1171034A - Diagnosis and treatment of autoimmune demyelinating diseases - Google Patents

Abstract

The present invention concerns the diagnosis and treatment of autoimmune demyelinating diseases, such as multiple sclerosis (MS), by means of a CLM-I agonist.

Description

Diagnosis and treatment of autoimmune demyelinating diseases

Technical Field

The present invention relates to the diagnosis and treatment of autoimmune demyelinating diseases, such as Multiple Sclerosis (MS).

Background

Bone marrow cells are the major effector cells in autoimmune demyelinating diseases (Barnett et al, Multiple scleroses 12, 121-. The CNS-infiltrating bone marrow population consists of resident microglia, macrophages, inflammatory dendritic cells, plasmacytoid dendritic cells and conventional dendritic cells. They have received particular attention due to the ability of myeloid Dendritic Cells (DCs) expressing MHCII and CD86 to reactivate antigen-specific T cells (deshopande et al, J Immunol (journal of immunology) 178, 6695-6699, 2007) and their involvement in epitope spreading leading to recurrent disease (Miller et al, J Immunol (journal of immunology) 178, 6695-6699, 2007). In addition to acting as antigen presenting cells, inflammatory DCs directly regulate the local extracellular environment by secreting proinflammatory cytokines and reactive oxygen intermediates, leading to progressive demyelination and axonal loss. Precursor cells of these TNF-and iNOS-producing dendritic cells, also known as TipDCs (Serbina et al, Immunity 19, 59-70, 2003), are inflammatory monocytes present in the circulation and recruited to areas of CNS inflammation. Conversion of inflammation to type II anti-inflammatory monocytes by glatiramerate (a drug approved for MS) leads to reversal of EAE severity (Weber et al, Nature Medicine 13, 935-943, 2007), further underscoring the important role of these myeloid cells in regulating disease severity.

Other negative regulators of CNS infiltrating myeloid cells have been previously identified. For example, TREM-2 expressed on both resident microglia and infiltrating myeloid cells plays an important role in resolution of CNS inflammation by phagocytosis of myelin debris (Piccio et al, European Journal of immunology 37, 1290. sup. 651301, 2007; Takahashi et al, PLoS Medicine 4, e124, 2007; Takahashi et al, The Journal of Experimental Medicine 201, 647. sup. 6572005, 2005). Similarly, IFNAR on bone marrow cells down-regulates inflammatory responses in the CNS (Prinz et al, Immunity 28, 675-686, 2008). However, none of the receptors is specific for inflammatory bone marrow-derived monocytes that home to the CNS.

CLM-1(MAIR-V, LMIR-3, DigR2) was identified in the search for bone marrow specific cell surface receptors important for down-regulation of bone marrow function. CLM-1 is part of the CMRF family, a multigene cluster on human chromosome 17, whose mouse ortholog is located on chromosome 11. All family members contain an extracellular IgV domain. Two family members in this cluster (CLM-1 and CLM-8) contain ITIM sequences in the intracellular domain, with the remaining members having charged residues in the transmembrane region, which can serve as recruitment signaling linkers. CLM-1, a murine ortholog of human CD300f (Clark et al, Trends in Immunology 30, 209-. Subsequent studies have shown that CLM-1 plays an inhibitory role in Fc-receptor mediated cellular responses (Alvarez-Errico et al, The Journal of Experimental Medicine 206, 595-606, 2004; Fujimoto et al, International Immunology 18, 1499-1508, 2006). To date, no biological role in autoimmune diseases has been described.

Summary of The Invention

The present invention is based, at least in part, on the identification of CLM-1, which is identified as a negative regulator of inflammatory DCs activity in the CNS by inhibiting the release of inflammatory cytokines and reactive oxygen species. Thus, in the present invention, CLM-1 was identified as a bone marrow-specific negative regulator of CNS inflammation and demyelination (demyelination).

In one aspect, the invention relates to a method of treating a demyelinating disease in a mammalian subject, the method comprising administering to the subject an effective amount of a CLM-1 agonist.

In another aspect, the invention relates to a pharmaceutical composition for the treatment of demyelinating diseases, comprising an effective amount of a CLM-1 agonist in admixture with a pharmaceutically acceptable excipient.

In another aspect, the invention relates to the use of an effective amount of a CLM-1 agonist in the manufacture of a medicament for the treatment of a demyelinating disease.

In another aspect, the invention relates to CLM-1 agonists for use in the treatment of demyelinating diseases.

In another aspect, the invention relates to a method for diagnosing a demyelinating disease, the method comprising detecting a defect in CLM-1 function.

In another aspect, the invention relates to a kit comprising a CLM-1 agonist and instructions for use for treating a demyelinating disease.

In all aspects, the invention specifically includes the following embodiments:

in one embodiment, the mammalian subject is a human.

In another embodiment, the demyelinating disease is a demyelinating autoimmune disease.

In another embodiment, the demyelinating autoimmune disease affects the Central Nervous System (CNS).

In another embodiment, the demyelinating autoimmune disease is selected from the group consisting of: multiple Sclerosis (MS), Relapsing Remitting MS (RRMS), primary and secondary progressive forms of MS (primary and secondary progressive forms of MS), progressive relapsing forms of MS (progressive compliance of MS), encephalomyelitis (encephalomyelitis), white matter encephalitis (leukoencephalitosis), transverse myelitis (transverse myelitis), neuromyelitis optica (neurosis), and optic neuritis (optic nerve).

In another embodiment, the demyelinating autoimmune disease is MS.

In various embodiments, the demyelinating autoimmune disease affects the peripheral nervous system, including, but not limited to, acute inflammatory demyelinating polyneuropathy (AIDP; Guillain-Barre syndrome); chronic inflammatory demyelinating polyneuropathy (chronic inflammatory demyelinating polyneuropathy); anti-MAG peripheral neuropathy; and Motor and Sensory Neuropathy (HMSN) (also known as Hereditary Sensory Motor Neuropathy (HSMN), or Peroneal Muscular Atrophy (Peroneal Muscular Atrophy), and progressive neuropathic Peroneal Muscular Atrophy (Charcot-Marie-Tooth Disease)).

In another embodiment, the CLM-1 agonist is an agonist anti-CLM-1 antibody.

Brief Description of Drawings

Figure 1 CLM-1 is expressed on inflammatory dendritic cells in CNS inflammatory lesions.

(A) Increased CLM-mRNA transcripts in the spinal cord at the most severe of the disease. (B) CLM-1 expression was absent on CNS-colonized CD11b + cells. (C) CLM-1 expression on CD11bCD11c + bone marrow cells. (D) CLM-1CD11c co-expresses DCs in CNS inflammatory lesions at the most severe of the disease (thoracic, dorsal horn). (E) DCs expressing CLM-1 express iNOS and TNF α. Values are expressed as mean + s.d. (D) The middle scale bar is 50 μm.

Figure 2 expression of CLM-1 on inflammatory monocytes and dendritic cells.

(A) CLM-1 at Cx3cr1^loCD11c⁺Ly6^hiPositive inflammatory monocytes, but not Cx3cr1^hiExpression on conventional DC precursors. (B) CLM-1 is expressed on radiation-sensitive bone marrow-derived cells, but not on radiation-resistant CNS-resident microglia. (C) CLM-1 at Cx3cr1^loInflammatory DCs but not Cx3cr1^hiExpression on microglia. (D) Cx3cr1 and CLM-1 were expressed on spinal cord sections (thoracic section) 14 days after immunization. Co-staining was observed at the periphery of the medullary membrane (arrows), whereas Cx3cr1hi microglia (arrow symbols) did not carry CLM-1. Scale bar: b (100 μm), D (50 μm).

FIG. 3. treatment with CLM-1 fusion protein or lack thereof results in increased EAE.

(A) The expression of CLM-1 protein was absent in bone marrow-derived DCs obtained from CLM-1 knockout (ko) mice (left panel). Similar levels of MHC II and CD86 were found on DCs obtained from the spinal cord where the disease was most severe (right panel). (B) Lack of CLM-1 staining, maintained morphology and similar inflammatory cell numbers in CLM-1wt compared to ko mice. (C) Increased disease severity in CLM-1ko mice or (D) CLM-1wt mice treated with CLM-1-Fc fusion proteins. (B) The scale bar in (1) is 50 μm.

FIG. 4.CLM-1 deficiency did not affect T cell priming.

(A) The proliferation and cytokine response of restimulated antigen-specific peripheral lymph node T cells were similar in CLM-1wt and ko mice. (B) In wild type recipients, T cells from CLM-1ko or wt donor mice induced similar disease (left panel). T cells from CLM-1wt donors induced increased disease severity in CLM-1ko recipients compared to CLM-1wt wild type recipients (right panel).

Figure 5 CLM-1 modulates the release of bone marrow-but not T cell-specific inflammatory mediators.

(A) There was no difference in the numbers of Th1, Th17 and regulatory T cells when MOG-responsive spinal cord T cells obtained from immunized CLM-1wt and ko mice were reactivated. (B) Increased DC activation in CLM-1wt and ko bone marrow cells obtained from CNS inflammatory injury. P < 0.01.

Figure 6 CLM-1 modulates autoimmune demyelination.

(A) Images of a superposition of CLM-1 positive cells and MOG positive myelin in CNS lesions. (B) And (C): there was increased demyelination in CLM-1ko compared to wt mice (shown by the white line marked area in B and quantified in C).

FIG. 7 amino acid sequences of mouse (SEQ ID NO: 1) and human (SEQ ID NO: 2) CLM-1 polypeptides.

Supplementary FIG. 1. strategy for targeted disruption of mouse Clm-1 gene.

ES cells were produced by homologous recombination in which Clm-1 exon-1 was replaced with a neomycin resistance gene. The structure of the targeting region of the Clm-1 gene is shown. E1 and E2 represent exon 1 and exon 2 of the Clm-1 gene. The probe locations (5 'and 3') used to screen ES clones by Southern blotting are shown.

Supplementary figure 2 CLM-1 did not affect T cell proliferation.

(A) T cells obtained from OVA transgenic T cells were incubated with bone marrow-derived dendritic cells obtained from CLM-1wt or ko mice in the presence of increasing concentrations of OVA peptide. (B) Mixed lymphocyte reaction. Bone marrow dendritic cells obtained from Balb/C background CLM-1wt or ko mice were incubated with varying ratios of T cells obtained from C57B1/6 background mice. Proliferation was reflected by H3 thymidine incorporation.

Supplemental figure 3 (a) Clm-1 did not affect the production of regulatory T lymphocytes in peripheral lymph nodes. (B) Clm-1 does not affect the polarization of T lymphocytes in the CNS (polization).

Detailed description of the preferred embodiments

I. Definition of

The terms "CLM-1" and "Cmrf-like molecule-1" (also known as MAIR-V, LMIR-3, DigR2 and IgSF13) are used interchangeably herein to refer to the native sequence mammalian CLM-1 receptor, specifically including, but not limited to, the mouse CLM-1 polypeptide SEQ ID NO: 1(NCBI CAM21607) and its human orthologs SEQ ID NO: 2(NCBI AAH28188, also known as CD300f, IREM1, IgSF13, 35-L5, and CMRF-35A5), and naturally occurring variants thereof. For further details and nomenclature see Clark et al, 2009, supra.

A "native sequence" polypeptide is a polypeptide having the same amino acid sequence as a polypeptide of natural origin (e.g., an ErbB receptor or ErbB ligand). The native sequence polypeptide may be isolated in nature, or may be produced recombinantly or synthetically. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring human polypeptide, a murine polypeptide, or a polypeptide from any other mammalian species.

The term "amino acid sequence variant" refers to a polypeptide having an amino acid sequence that differs to some extent from a native sequence polypeptide. Typically, amino acid sequence variants are at least about 70% homologous to at least one receptor binding domain of a native ErbB ligand or to at least one ligand binding domain of a native ErbB receptor, preferably they are at least about 80%, more preferably at least about 90% homologous to the receptor or ligand binding domain. The amino acid sequence variants have substitutions, deletions, and/or insertions at specific positions in the amino acid sequence of the native amino acid sequence.

"homology" is defined as the percentage of residues that are identical in amino acid sequence variants after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent homology. Methods and computer programs for alignment are well known in the art. One such computer program is "Align 2", owned by Genentech, Inc (key taycota biotechnology corporation), filed on 12/10 of 1991 by the U.S. copyright office of washington, dc 20559.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be produced by a method originally described by Kohler et al, Nature, 256: 495(1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Such "monoclonal antibodies" may also be used, for example, in Clackson et al, Nature (Nature), 352: 624-: 581-597(1991) from phage antibody libraries.

Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA) 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., cynomolgus Monkey (Old World Monkey), ape, etc.) and human constant region sequences.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

An "intact" antibody is one that comprises an antigen-binding variable region and a light chain constant domain (C)_L) And heavy chain constant domain, C_H1，C_H2 and C_H3. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include: a C1q bond; complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like

Depending on the amino acid sequence of their heavy chain constant domains, whole antibodies can be assigned to different "classes". There are five main types of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA 2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to cell-mediated reactions in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annual assessment of immunity (annu. rev. immunol.) 9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as the assays described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-.

"human effector cells" refer to leukocytes which express one or more FcRs and perform effector functions. Preferably, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; among them, PBMCs and NK cells are preferable. As described herein, effector cells can be isolated from their natural source, e.g., from blood or PBMCs.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB comprises in its cytoplasmic domain an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see review M in Da ё ron, Annu. Rev. Immunol.) 15: 203-234 (1997). For a review of fcrs see ravatch and Kinet, annual assessment of immunity (annu. rev. immunol.) 9: 457-92 (1991); capel et al, immunization methods (immunoassays) 4: 25-34 (1994); and de Haas et al, journal of laboratory clinical medicine (j.lab.clin.med.) 126: 330-41(1995). The term "FcR" herein encompasses other fcrs, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, journal of immunology (j.immunol.) 117: 587(1976) and Kim et al, journal of immunology (j.immunol.) 24: 249 (1994)).

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) that is complexed with a relevant antigen. To assess complement activation, CDC assays can be performed, e.g., as in Gazzano-Santoro et al, journal of immunological methods (j.immunol.methods) 202: 163 (1996).

A "natural antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at one end (V)_H) Followed by a plurality of constant domains. Each light chain has a variable domain at one end (V)_L) And has a constant domain at its other end. The light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, which mostly adopt a β -sheet configuration, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and together with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable regions typically comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain, and residues 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain; Kabat et al, protein Sequences of Immunological Interest (Sequences of Proteins of Immunological Interest), 5 th edition, Public Health Service (Public Health Service), National institutes of Health (National Institute of Health, Bethesda, MD. (1991)) and/or those from the "hypervariable loops" (e.g., residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain, and residues 26-32(H1), 53-55 (H6355) and 96-96 (H6332) and H3 (H2) in the heavy chain variable domain), j.mol.biol. (journal of molecular biology) 196: 901-917(1987)). "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments (each with a single antigen-binding site), and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition site and the antigen binding site. The region consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in close, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact so as to be at V_H-V_LThe dimer defines an antigen binding site on the surface. In general, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, but with less affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name given herein for Fab', in which the cysteine residues of the constant domains have at least one free thiol group. F (ab')₂Antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

An antibody "light chain" from any vertebrate species can be assigned to one of two distinctly different types, termed kappa (κ) and lambda (λ), based on the amino acid sequences of its constant domains.

"Single chain Fv" or "scFv" antibody fragments comprise antibody V_HAnd V_LDomains, wherein the domains are present in a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains to enable the scFv to form the desired antigen binding structure. For reviews on scFv see Pl ü ckthun, The Pharmacology of Monoclonal Antibodies (Monoclonal antibody Pharmacology), Vol 113, Rosenburg and Moore eds, Springer-Verlag, New York, p.269-315 (1994). anti-ErbB 2 antibody scFv fragments are described in WO 93/16185; U.S. patent nos. 5,571,894; and U.S. Pat. No. 5,587,458.

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are comprised in the same polypeptide chain (V)_H-V_L) Neutralizing variable light chain domain (V)_L) Linked variable heavy chain domains (V)_H). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are caused to pair with complementary domains in the other chain, and two antigen binding sites are created. Diabodies are described, for example, in EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. AcadUsa (proceedings of the national academy of sciences usa), 90: 6444- > 6448 (1993).

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that comprise minimal sequences derived from non-human immunoglobulins. To the greatest extent, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all or at least one, and typically two, such variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature (Nature) 332: 323-329 (1988); and Presta, curr.op.struct.biol. (new view of structural biology) 2: 593-596(1992).

An "isolated" antibody is one that has been identified and separated from and/or recovered from a component of its natural environment. Contaminant components of the natural environment of an antibody refer to substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotating cup sequencer (spotting cup sequencer), as determined by the Lowry method, of more than 95% by weight of the antibody, and most preferably more than 99% by weight of the antibody, or (3) to homogeneity by using coomassie blue or, preferably, silver stained SDS-PAGE under reducing or non-reducing conditions. An isolated antibody includes an antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

An antibody that "binds" an antigen of interest is one that is capable of binding the antigen with sufficient affinity such that the antibody can be used as a therapeutic agent to target cells expressing the antigen.

The term "demyelinating disease" is used herein to refer to any neurological disease in which the myelin sheath of neurons is damaged. This definition includes diseases that affect the integrity of oligodendrocytes and their ability to produce and maintain myelin, and diseases that directly damage myelin. The disease disturbs transduction of the myelinated white matter pathway and produces a wide range of motor, sensory, and cognitive dysfunctions, including impairment of sensory, motor, cognitive, and/or other functions, depending on which nerves are involved, including Central Nervous System (CNS) nerves and peripheral nerves.

An "autoimmune disease" herein is a disease or disorder caused by and directed to or resulting from an individual's own tissue or a co-segregation or manifestation thereof. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis (psoriasis), dermatitis (dermatitis), including atopic dermatitis (atopic dermatitis); chronic idiopathic urticaria (chronic idiopathic urticaria) including chronic autoimmune urticaria (chronic autoimmune urticaria), polymyositis (polymyositis)/dermatomyositis (dermatomyositis), toxic epidermal necrolysis (toxic epidermal necrolysis), systemic scleroderma (systemic scleroderma) and scleroderma (scleroderma), IBD (inflammatory bowel disease, IBD) related reactions (Crohn's disease), ulcerative colitis (ulcerative colitis), and IBD with paraderma gangrenosum (malignant epithelial pyoderma), erythema nodosum (erythronodosum), primary sclerosing bile duct (primary scleroderma and/or scleroderma), respiratory tract (inflammatory bowel disease), acute respiratory syndrome (acute respiratory distress syndrome), acute respiratory distress syndrome (acute respiratory distress syndrome), chronic allergic rhinitis syndrome (acute respiratory distress syndrome), chronic obstructive pulmonary distress syndrome), encephalitis (encephalitis), such as Rasmussen's encephalitis (Rasmussen's encephalitis), uveitis (uveitis), colitis (coitis), such as microscopic colitis (microscopical colitis) and collagenous colitis (collagenous colitis), glomerulonephritis (glomerization, GN), such as membranous GN (membranous), idiopathic membranous GN (idiophatic membrane GN), membranous proliferative GN (membranous proliferative GN, MPGN), which includes types I and II, and rapidly progressive GN (systemic proliferative GN), allergic disorders (allergic disorders), eczema (eczema), asthma (infiltrative), disorders involving T cells and chronic inflammatory reactions (systemic inflammation of T cells), systemic lupus erythematosus (systemic sclerosis), systemic lupus erythematosus (systemic lupus erythematosus), systemic lupus erythematosus (systemic sclerosis), systemic lupus erythematosus, systemic lupus erythematosus, lupus, lupus (lupis) (including nephritis (nephritis), cerebritis (cerebritis), childhood (peditrics) lupus, non-renal (non-renal) lupus, discoid (discoid) lupus, alopecia (alopecia) lupus), juvenile onset diabetes (jungle onset diabetes), Multiple Sclerosis (MS), such as spinal-optic MS (spino-optic MS), allergic encephalomyelitis (allogenic encephalyitis), immune responses associated with acute and delayed hypersensitivity reactions (acute and delayed hypersensitivity) mediated by cytokines and T lymphocytes, tuberculosis (tuberculosis), sarcoidosis (sarcoidosis), granulomatosis (granulomatosis), including Wegener' granulomatosis), vasculitis (vasculitis) (including vascular hypertension), vasculitis (vasculitis) (including megavasculitis (vasculitis), polycythemitis (vasculitis (macroangiopathy), polycythemia (vasculitis), and polycythemia (vasculitis) (including rheumatoid arthritis), medium vascular vasculitis (Medium vascular vasculitis), including Kawasaki's Disease and Polyarteritis Nodosa (Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), aplastic anemia (autoimmune anemia), Coombs positive anemia (Coombs specific anemia), donning-cloth anemia (diamondd Blackfan anemia), immune hemolytic anemia (autoimmune hemolytic anemia), including autoimmune hemolytic anemia (autoimmune hemolytic anemia, AIHA), pernicious anemia (malignant anemia), simple regenerative disorder (pure vascular anemia, PRCA), Factor VIII deficiency (pathogenic Factor VIII), reduced erythrocyte leucocyte (hemophilia), reduced erythrocyte leucopenia (hemophilia), diseases involving leukocyte blood cell extravasation (diseases involving leukocyte depletion Syndrome), CNS inflammatory disorders, multiple organ injury Syndrome (multiple organ injury Syndrome), myasthenia gravis (myasthenia gravis), antigen-antibody complex-mediated diseases (anti-antigen-mediated diseases), anti-glomerular basement membrane disease (anti-glomerular basal membrane disease), anti-phospholipid antibody Syndrome (anti-phospholipid antibody Syndrome), allergic neuritis (allergic neuronitis), Bechet disease, casseman's Syndrome, Goodpasture's Syndrome, labert-eaton Syndrome (labyrinthine-mediated Syndrome), labyrinthine-eaton-eathesis Syndrome (labyrinthine-induced Syndrome), labyrinthine-eaton Syndrome (labyrinthine-synstellite Syndrome), labyrinthine-eaton-syndet Syndrome (labyrinthine-Syndrome), labyrinthine-Syndrome (labyrinthine-Syndrome), graft-Syndrome (Sjongshiandrein-induced Syndrome), and autoimmune Syndrome (autoimmune disease), systemic inflammatory Syndrome (autoimmune Syndrome), systemic inflammatory Syndrome, autoimmune diseases including autoimmune Syndrome, autoimmune diseases, autoimmune Syndrome (autoimmune diseases, autoimmune, Pretreatment of IgA deposition in tissues, and rejection by kidney transplantation, liver transplantation, intestine transplantation, heart transplantation, etc., Graft Versus Host Disease (GVHD), bullous pemphigoid (pemphigoid), pemphigus (pemphigus), including pemphigus vulgaris (vulgaris), epifoliate (foliaceus), and pemphigoid (pemphigoid-mucosae), autoimmune polyendocrinopathy (autoimmune neuronopathies), Retention's disease, Stiff-mandrome syndrome (stiff-mandrome), immune complex nephritis (immune complex nephritis), IgM polyneuropathy (IgM) or neuro-mediated neuropathy (IgM), thrombocytopenic purpura (thrombocytopenia), e.g., thrombocytopenia), thrombocytopenia (thrombocytopenia), thrombopenia (idiopathic thrombocytopenia), including autoimmune thrombocytopenia (autoimmune disease of the liver and kidney), autoimmune testicular and ovarian diseases (autoimmune diseases of the liver and ovary), including autoimmune orchitis and oophoritis (ovarian), primary hypothyroidism (primary hypothyroidism); autoimmune endocrinopathies (autoimmune thyroiditis), including autoimmune thyroiditis (autoimmune thyroiditis), chronic thyroiditis (chronic thyroiditis) (Hashimoto's thyroiditis), subacute thyroiditis (subacute thyroiditis), idiopathic hypothyroidism (idiopathy hypothyroidism), Addison's disease, Graves 'disease, autoimmune polyglandular syndrome (autoimmune polyglandular syndrome) (or polyglandular endocrine syndrome), type I diabetes, also known as insulin-dependent diabetes mellitus (insulin-dependent diabetes mellitus, including Children's syndrome, Sheedm); autoimmune hepatitis (autoimmune hepatitis), Lymphoid interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplantable) vsNSIP in contrast to NSIP, Guillain-Barre Syndrome (Guillain-Barre Syndrome), Beger's disease (IgA nephropathy), primary biliary cirrhosis (primary biliarlity disorders), sprue (celiac disease) (gluteal enteropathy), refractory sprue with co-separation of herpetiformis (cervical intestinal disease), sprue with co-separation of herpetiformis (coronary artery with-regression), cryoglobulinemia (cryoglobulinemia), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), autoimmune encephalopathy (autoimmune encephalopathy), ocular clonus myoclonus syndrome (OMS), polychondritis (polychondritis) such as refractory polychondritis (recurrent polychondritis), alveolar proteinosis (pulmonary alveolar amyloidosis), amyloidosis (amyloidosis), giant cell hepatitis (giant cell hepatitis), scleritis (scleritis), monoclonal propanaminopathy of undetermined/unknown importance (monoclonal gamma-associated neuropathy of uncertain/unknown importance, MGUS), peripheral neuropathy (peripheral neuropathy), paraneoplastic syndrome (paraneoplastic syndrome), channelopathies (channelopathies) such as epilepsy (epilepsy), cardiac arrhythmia (cardiac arrhythmia), migraine (depression), depression (depression), depression (depression ), depression (depression; autism (autism), inflammatory myopathy (inflammation myopathy), and Focal Segmental Glomerulosclerosis (FSGS).

The terms "disease characterized by autoimmune demyelination" and "demyelinating autoimmune disease" are used interchangeably and refer to a demyelinating disease caused, at least in part, by an autoimmune reaction. Demyelinating autoimmune diseases include recurrent or chronic progressive demyelinating diseases such as Multiple Sclerosis (MS) and variants thereof, and monophasic demyelinating diseases such as optic neuritis (optic neuritis), acute disseminated encephalomyelitis (acute disseminated encephalomyelitis), and transverse myelitis (transverse myelitis). Demyelinating autoimmune diseases of the Central Nervous System (CNS) include, but are not limited to, MS and MS variants, such as Relapsing Remitting MS (RRMS) and primary and secondary progressive forms, and progressive relapsing forms of MS, encephalomyelitis, white matter encephalitis (leukoencephalitis), transverse myelitis, neuromyelitis optica (Devic's disease), and optic neuritis (opthaleuritis). Demyelinating autoimmune diseases affecting the peripheral nervous system include, for example, acute inflammatory demyelinating polyneuropathy (AIDP; Guillain-Barre syndrome); chronic inflammatory demyelinating polyneuropathy (chronic inflammatory demyelinating polyneuropathy); anti-MAG peripheral neuropathy (anti-MAG periphytol neuropathy); and Motor and Sensorineural (HMSN), also known as genetic sensorineural Neuropathy (HSMN), or Peroneal muscular atrophy (Peroneal muscular atrophy), or progressive neuropathic Peroneal muscular atrophy (Charcot-Marie-Tooth Disease).

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented. Thus, the mammal to be treated herein may have been diagnosed with the disorder or may be predisposed or predisposed to the disorder. For example, prophylactic treatment includes a clinical form that prevents complete progression, or a more severe form of disease, such as preventing the progression of MS to relapsing remitting MS (rrms). Therapeutic treatment may be aimed at slowing the progression of the disease, reducing the frequency of disease onset (attacks), restoring function after an attack, preventing new attacks, and preventing or slowing the development of disabilities associated with or caused by the disorder.

The term "CLM-1 agonist" is used herein in the broadest sense and includes any molecule that partially or completely enhances, stimulates or activates one or more CLM-1 biological activities in vitro, in situ or in vivo. For example, the agonist may function to partially or fully enhance, stimulate or activate one or more CLM-1 biological activities in vitro, in situ, or in vivo as a result of direct binding to CLM-resulting in receptor activation or signal transduction. The agonist may also function indirectly by, for example, stimulating another effector molecule which then causes CLM-1 activation or signal transduction, partially or fully enhancing, stimulating or activating one or more CLM-1 biological activities in vitro, in situ, or in vivo. The biological activity herein is the negative regulation of demyelinating diseases, such as the demyelinating autoimmune diseases defined above. Agonists include in particular CLM-1 ligands and agonist antibodies against CLM-1.

"mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, non-human higher primates, domestic and farm animals, and zoo, sports, or pet animals such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or condition in a mammal. In this case, a therapeutically effective amount is an amount of CLM-1 agonist effective to treat (including prevent) a demyelinating disease (a demyelinating autoimmune disease as defined hereinbefore).

"liposomes" are vesicles composed of various types of lipids, phospholipids and/or surfactants useful for delivering drugs (such as the anti-ErbB 2 antibodies disclosed herein, and optionally chemotherapeutic agents) to mammals. The components of liposomes are typically arranged in a bilayer structure, similar to the lipid arrangement of biological membranes.

The term "package insert" is used to refer to instructions for use, typically contained in commercial packaging for a therapeutic product, that contain information about the indication, application, dosage, administration, contraindications, and/or warnings concerning the use of the therapeutic product.

Detailed description of the invention

Multiple Sclerosis (MS) and its latent equivalent Experimental Autoimmune Encephalomyelitis (EAE) are characterized by perivascular inflammation and demyelination. Bone marrow cells derived from circulating progenitor cells are the major component of inflammatory exudates and are thought to constitute the ultimate effector cells responsible for cytokine production, demyelination, axonal injury, and motor dysfunction. Little is understood about how to modulate the cytotoxic activity of these bone marrow cells. The present invention is based, at least in part, on the identification of Cmrf-like molecule-1 (CLM-1) as a negative modulator of autoimmune demyelination. Upon immunization of mice with MOG peptide, CLM-1 was expressed on inflammatory monocytes in peripheral blood and on inflammatory dendritic cells present in areas of CNS demyelination. The lack of CLM-1 on CNS infiltrating inflammatory dendritic cells results in significantly increased nitric oxide and proinflammatory cytokine production, as well as increased clinical scores for axonal demyelination and deterioration, while T cell responses remain unaffected. Thus, CLM-1 is identified herein as a negative regulator of bone marrow cell activation and autoimmune demyelination.

Bone marrow cells are the major effector cells in autoimmune demyelinating diseases (Barnett et al, Multiple scleroses (Multiple Sclerosis) (Houndmolls, Basingstoke, England)12, 121-. The CNS-infiltrating bone marrow population consists of resident microglia, macrophages, inflammatory dendritic cells, plasmacytoid dendritic cells and conventional dendritic cells. Bone marrow Dendritic Cells (DCs) expressing MHCII and CD86 have received particular attention due to their ability to reactivate antigen-specific T cells (Deshpanded et al, J Immunol (J Immunol) 178, 6695-. In addition to acting as antigen presenting cells, inflammatory DCs directly regulate the local extracellular environment by secreting proinflammatory cytokines and reactive oxygen intermediates, leading to progressive demyelination and axonal loss. Precursor cells of these TNF-and iNOS-producing dendritic cells, also known as TipDCs (Serbina et al, Immunity 19, 59-70, 2003), are inflammatory monocytes present in the circulation and recruited to areas of CNS inflammation. Conversion of inflammation to type II anti-inflammatory monocytes by glatiramer acetate, a drug approved for MS, resulted in reversal of EAE severity (Weber et al, Nature Medicine 13, 935-943, 2007), further underscoring the important role of these myeloid cells in regulating disease severity.

Other negative regulators of CNS infiltrating myeloid cells have been previously identified. For example, TREM-2 expressed on both resident microglia and infiltrating myeloid cells plays an important role in resolution of CNS inflammation by phagocytosis of myelin debris (Piccio et al, European Journal of immunology 37, 1290-. Similarly, IFNAR on bone marrow cells down-regulates inflammatory responses in the CNS (Prinz et al, Immunity 28, 675-686, 2008). However, none of the receptors is specific for inflammatory bone marrow-derived monocytes that home to the CNS.

CLM-1(MAIR-V, LMIR-3, DigR2) was identified in the search for bone marrow specific cell surface receptors important for down-regulation of bone marrow function. CLM-1 is part of the CMRF family, a multigene cluster on human chromosome 17, whose mouse ortholog is located on chromosome 11. All family members contain an extracellular IgV domain. Two family members in this cluster (CLM-1 and CLM-8) contain ITIM sequences in the intracellular domain, with the remaining members having charged residues in the transmembrane region, which can serve as recruitment signaling linkers. CLM-1(SEQ ID NO: 1), a murine ortholog of human CD300f (SEQ ID NO: 2) (Clark et al, Trends in Immunology 30, 209. sup. 217, 2009), was originally described as a negative regulator of osteo-mitogenesis (osteoprogenitor) (Chung et al, J.Immunol (J.Immunol) 171, 6541. sup. 6548, 2003). Subsequent studies have shown that CLM-1 plays an inhibitory role in Fc-receptor mediated cellular responses (Alvarez-Errico et al, 2004; Fujimoto et al, 2006). To date, no biological role in autoimmune diseases has been described. In the present invention, we identified CLM-1 as a negative regulator of inflammatory DCs activity in the CNS by inhibiting the release of inflammatory cytokines and reactive oxygen species. Thus, the present study identified CLM-1 as a bone marrow-specific negative regulator of CNS inflammation and demyelination.

The present invention relates to methods of diagnosing and treating demyelinating diseases, such as demyelinating autoimmune diseases, with CLM-1 antagonists.

In a specific embodiment, the CLM-1 agonist is an agonist antibody directed against CLM-1.

Antibodies

Antibodies of the invention include anti-CLM-1 antibodies or antigen-binding fragments of CLM-1, or other antibodies described herein. Exemplary antibodies include, for example, polyclonal antibodies, monoclonal antibodies, humanized antibodies, fragments, multispecific antibodies, heteroconjugate (heteroconjugate) antibodies, multivalent antibodies, effector function antibodies, and the like. In a particular embodiment of the invention, the antibody is an agonist antibody.

Polyclonal antibodies

The antibodies of the invention may include polyclonal antibodies. Methods for preparing polyclonal antibodies are known to the skilled worker. For example, polyclonal antibodies against CLM-1 are generated in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, for example, using a bifunctional or derivatizing agent (e.g., maleimidobenzoyl sulfosuccinimidyl ester (conjugated through a cysteine residue), N-hydroxysuccinimidylAmines (via lysine residues), glutaraldehyde, succinic anhydride, or SOCl₂) The antigen may be conjugated to a keyhole limpetHemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

Animals are immunized against CLM-1, immunogenic conjugates or derivatives by mixing, for example, 100 or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals, and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (pateau). Typically, animals are boosted with conjugates of the same antigen but conjugated to different proteins and/or conjugated through different cross-linking agents. Conjugates can also be prepared in recombinant cell culture as protein fusions. Also, a coagulant (such as alum) is suitably used to enhance the immune response.

Monoclonal antibodies

Monoclonal antibodies may be used as originally produced by Kohler et al, Nature (Nature) 256: 495(1975), or can be prepared by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal (such as a hamster or cynomolgus monkey) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103, (academic Press, 1986)).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Typical myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Preferred myeloma Cell lines of these are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, Calif., USA) of San Diego, Calif., and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection of Rockwell, Md.USA, Md.. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the Production of human Monoclonal antibodies (Kozbor, J.Immunol.133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications (Monoclonal Antibody Production technologies and Applications), pp.51-63, (Marcel Dekker, Inc., New York, 1987)).

The medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against CLM-1. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Munson and Pollard, anal. biochem. (journal of biochemistry) 107: 220(1980) by Scatchard analysis (Scatchard analysis).

After identification of hybridoma cells producing Antibodies with the desired specificity, affinity and/or activity, the clones can be subcloned by limiting dilution methods and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification methods such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. patent No. 4,816,567. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells may serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin protein, such as an escherichia coli (e.coli) cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of monoclonal antibodies in the recombinant host cell. Recombinant production of antibodies will be described in more detail below.

In another embodiment, the method may be selected from the group consisting of using McCafferty et al, Nature 348: 552 (1990) and isolating antibodies or antibody fragments from antibody phage libraries. Clackson et al, Nature 352: 624-: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of human antibodies of high affinity (nM range) by chain shuffling (Marks et al, Bio/Technology (Bio/Technology) 10: 779-. As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified, for example, by replacing the homologous murine sequences with the human heavy and light chain constant domain coding sequences (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA) 81: 6851(1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the non-immunoglobulin polypeptide coding sequence.

Typically, the non-immunoglobulin polypeptides are used to replace the constant domains of an antibody, or they are used to replace the variable domains of one antigen binding site of an antibody, to produce a chimeric bivalent antibody comprising one antigen binding site with specificity for one antigen and another antigen binding site with specificity for a different antigen.

Humanized and human antibodies

The antibodies of the invention may include humanized antibodies or human antibodies. Humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences (Jones et al, Nature, 321: 522-525 (1986); Riechmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than the entire human variable domain is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from analogous sites in rodent antibodies.

The choice of human variable domains (both light and heavy) for making humanized antibodies is very important for reducing antigenicity. The variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences according to the so-called "best-fit" method. The human sequences closest to the rodent sequences were accepted as the human Framework (FR) for the humanized antibody (Sims et al, J.Immunol. (J.Immunol.); Chothia et al, J.mol.biol. (J.Mol.Mobiol.) (J.Mobiol., 196: 901 (1987)). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA), 89: 4285(1992), Presta et al, J.Immunol., 151: 2623 (1993)).

More importantly, antibodies should be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to typical methods, humanized antibodies are prepared by a method of analyzing the parent sequence and various conceptual humanized products using three-dimensional modeling of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. A computer program is available which exemplifies and displays the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues from the recipient and import sequences can be selected and combined to achieve desired antibody characteristics, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in affecting antigen binding.

Alternatively, transgenic animals (e.g., mice) can now be produced that are capable of producing a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human germline immunoglobulin gene array into the germline mutant mouse results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa (proceedings of the national academy of sciences usa), 90: 2551 (1993); jakobovits et al, Nature (Nature), 362: 255-258 (1993); bruggemann et al, Yeast in immunity, 7: 33 (1993); and Nature 355 of Duchosal et al: 258(1992). Human antibodies can also be derived from phage display libraries (Hoogenboom et al, J.mol.biol. (J.Mol., molecular biol., 227: 381 (1991); Marks et al, J.Mol.biol. (J.Mol., molecular biol., 222: 581-597 (1991); Vaughan et al, Nature Biotech (Nature Biotechnology) 14: 309 (1996)).

Human antibodies can also be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol. (J.Mol., molecular biol., 227: 381 (1991); Marks et al, J.mol.biol. (J.Mol., molecular biol., 222: 581 (1991)). According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle comprises a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, reviewed, for example, in Johnson, K S, and Chiswell, D j., Current view of Structural Biology (Current Opinion in Structural Biology) 3: 564-571(1993). Several sources of V gene segments are available for phage display. For example, Clackson et al, Nature 352: 624-628(1991) a large number of different anti-oxazolone antibodies were isolated from a random combinatorial library of small V genes derived from the spleen of immunized mice. For example, by essentially following Marks et al, journal of molecular biology (j.mol.biol.) 222: 581-597(1991) or Griffith et al, EMBO J.12: 725-734(1993) allows the construction of V gene repertoires from non-immunized human donors and the isolation of antibodies against a large number of different antigens, including self-antigens. See also U.S. Pat. nos. 5,565,332 and 5,573,905. The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Monoclonal Antibodies with Cancer Therapy), Alan R.Liss, p.77 (1985) and Boerner et al, J.Immunol., 147 (1): 86-95 (1991)). Human antibodies can also be generated from in vitro activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

Antibody fragments

The invention also includes antibody fragments. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117(1992) and Brennan et al, Science 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli (E.coli) and chemically coupled to form F (ab'). sub.2 fragments (Carter et al, Bio/Technology 10: 163-167 (1992)). According to another approach, the F (ab'). sub.2 fragment can be isolated directly from the recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent nos. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only types with intact binding sites, lacking constant regions; as such, they are suitable for reducing non-specific binding when used in vivo. sFv fusion proteins can be constructed to produce fusion of an effector protein at the amino or carboxy terminus of an sFv. See Antibody Engineering (Antibody Engineering), eds. Borebaeck, supra. Antibody fragments may also be "linear antibodies," for example as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Multispecific (e.g., bispecific) antibodies

Antibodies of the invention also include, for example, multispecific antibodies that have binding specificities for at least two different antigens. Although such molecules will typically bind only two antigens (i.e., bispecific antibodies, BsAbs), when used herein, this expression encompasses antibodies with additional specificity, such as trispecific antibodies.

Methods for making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al, EMBO J.10: 3655-3659(1991).

According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably, the fusion has an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, a first heavy chain constant region (CH1) is present in at least one of the fusions that comprises the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into different expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide the desired optimal yield of bispecific antibody, this provides greater flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in the same ratio leads to high yields or when the ratio is of no particular significance.

In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combinations. The method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology 121: 210(1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the antibody constant domain CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing heterodimer production over other unwanted end products, such as homodimers.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229: 81(1985) describes the proteolytic cleavage of intact antibodies to yield F (ab')₂A method for fragmenting. These fragments are reduced in the presence of the dithiol complexing agent sodium arseniteTo stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to the Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Various techniques for the direct production and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, journal of immunology (j. immunol.), 148 (5): 1547-1553(1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Hollinger et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa), 90: 6444-. The fragment comprises a light chain variable domain (V) joined by a linker_L) A linked heavy chain variable domain (v.sub.h), the linker being too short to allow pairing between the two domains on the same chain. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for generating bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, journal of immunology (j. immunol.), 152: 5368(1994).

Antibodies with more than two titers are expected. For example, trispecific antibodies can be prepared. Tutt et al, journal of immunology (j. immunol.) 147: 60(1991).

Heteroconjugate antibodies

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies, which are antibodies of the invention. For example, one antibody of the heteroconjugate can be conjugated to avidin and the other to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with various crosslinking techniques.

Multivalent antibodies

Antibodies of the invention include multivalent antibodies. Multivalent antibodies may be internalized (and/or catabolized) by cells expressing the antigen to which the antibody binds faster than bivalent antibodies. The antibodies of the invention can be multivalent antibodies (which are outside the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibody. A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, a polypeptide chain can comprise VD1- (X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, a polypeptide chain can comprise: VH-CH 1-flexible linker-VH-CH 1-Fc region chain; or VH-CH1-VH-CH1-Fc domain chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody herein may comprise, for example, about two to about eight light chain variable domain polypeptides. Light chain variable contemplated hereinThe domain polypeptide comprises a light chain variable domain, and optionally further comprises a CL domain.

Effector function engineering

It may be desirable to modify the antibodies of the invention in terms of effector function, e.g., to enhance the efficacy of the antibodies in treating disease. For example, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody so produced may have improved internalization capability. See Caron et al, j.exp.med (journal of experimental medicine) 176: 1191-1195(1992) and shop, B. J.Immunol. 148: 2918-2922(1992). To extend the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment) as described, for example, in U.S. patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., igg.sub.1, igg.sub.2, igg.sub.3, or igg.sub.4) that is responsible for extending the serum half-life of the IgG molecule in vivo.

Other antibody modifications

Other modifications of the antibody are contemplated by the invention. For example, the antibody may be linked to a variety of non-proteinaceous polymers, for example, to polyethylene glycol, polypropylene glycol, polyoxyalkenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization methods (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microcapsules, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical Sciences, 16 th edition, Oslo, A., Ed., (1980).

Liposomes and nanoparticles

The CLM-1 antibodies of the invention may also be formulated as immunoliposomes. Liposomes comprising the polypeptide are prepared by methods known in the art, such as those described in Epstein et al, proc.natl.acad.sci.usa (proceedings of the national academy of sciences usa), 82: 3688 (1985); hwang et al, proc.natl.acad.sci.usa (proceedings of the national academy of sciences usa), 77: 4030 (1980); and U.S. patent nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Pat. No. 5,013,556. In general, the formulation and use of liposomes is known to those skilled in the art.

Particularly useful liposomes can be produced by reverse phase evaporation, having a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter defining a pore size to produce liposomes having a desired diameter. The polypeptides of the invention can be conjugated to liposomes by disulfide exchange reactions, as described in Martin et al j.biol.chem. (journal of biochemistry) 257: 286-. Nanoparticles or nanocapsules may also be used to entrap the polypeptides of the invention. In one embodiment, biodegradable polyalkyl-cyanoacrylate nanoparticles may be used for the polypeptides of the present invention.

Further details of the invention are illustrated by the following non-limiting examples. All publications cited in this specification are expressly incorporated herein by reference.

Examples

Materials and methods

Animals all animals were maintained under sterile, pathogen-free conditions, and animal experiments were approved by the Genentech institutional animal care and use committee. To generate Clm-1 Knockout (KO) mice, they will contain a neomycin resistance gene (Neo)^r) Electroporating the linearized targeting vector of (a) into C57Bl/6 derived C2 Embryonic Stem (ES) cells. Selection of neomycin resistant ES clones for Southern blot analysis of homologous recombination (supplementary figure)). Clm-1 exon 1 was successfully replaced by Neo^rThe gene-replaced ES clones were injected into C57BL/6 embryonic cells and subsequently transferred into pseudopregnant females to produce chimeric offspring. The chimeras were crossed with C57BL/6 mice to produce hybrids. Germline-transmitted hybrids with the targeted allele were backcrossed with C57BL/6 for at least 10 generations, followed by heterohybridization to produce Clm-1 wild-type (WT) and KO mice. C57BL/6 (in CD45.1 or CD45.2 isogenic background) mice from Jackson laboratory. Cx3cr1^gfp/+The C57BL/6 reporter mice were bred and maintained in a pathogen-free animal facility from Genentech. All animals were used at 8-12 weeks of age, except for the CD45.1/CD45.2 bone marrow chimera experiment, in which 6 weeks of age C57BL/6(CD45.1) was used as the bone marrow recipient. All experimental procedures were approved by the Genentech company institutional animal care and use committee.

Antibodies and recombinant proteins the following antibodies were purchased from BD Biosciences (BD Biosciences): anti-Fc γ RIII/II (CD32/16, clone 2.4G 2); PE-, APC-, APC-Cy 7-labeled anti-CD 11b (M1/70); biotin-, PE-, APC-labeled anti-CD 11c (HL 3); PE-, APC-labeled anti-CD 4(GK 1.5); APC-labeled anti-CD 3(145-2C 11); PE-Cy 7-labeled anti-B220 (RA3-6B 2); PE-labeled anti-I-A/I-E (M5/114.15.2); biotin-, PE-labeled anti-CD 86(GL 1); APC-Cy 7-labeled anti-Gr-1 (RB6-8C 5); PE-labeled anti-CD 45.1 (A20); biotin-, FITC-labeled anti-CD 45.2 (104); alexa Fluor 488-labeled anti-FoxP 3(MF 23); PE-labeled anti-IL-17 (TC11-18H 10); FITC-labeled anti-IFN □ (XMG 1.2); FITC-, PE-labeled anti-TNF α (MP6-XT 22); biotin-, PE-, PerCP-Cy5.5-labeled anti-CD 45 (30-F11); polyclonal rabbit anti-iNOS type II antibodies. The following antibodies were purchased from eBioscience: pacific blue-labeled anti-CD 11b (M1/70); PE-Cy 7-labeled anti-CD 11c (N418); PE-Cy 5-labeled anti-I-A/I-E (M5/114.15.2); APC-AlexaFluor 750-labeled anti-F4/80 (BM 8). Streptavidin Pacific Orange was purchased from Invitrogen. PE-labeled donkey anti-rabbit IgG and Cy 3-labeled anti-hamster IgG were purchased from jacksonn immunoresearch. Monoclonal anti-actin antibody (AC-40) was purchased from Sigma-Aldrich. To generate the murine Clm-1-Fc fusion protein, of murine Clm-1The extracellular domain (ECD) was cloned into a modified pRK5 expression vector encoding a murine IgG1Fc fragment. The expression vector was transfected into CHO cells and Clm-1-Fc fusion protein contained in the cell culture supernatant was purified by protein A affinity chromatography followed by Superdex 200 gel filtration. The properties of the purified protein were verified by mass spectrometry with endotoxin levels < 0.05 EU/mg. A murine anti-gp 120 antibody (IgG1) was used as a control. Monoclonal antibodies directed against the ECD of murine Clm-1 were generated by immunization of the hamster of Yamenia with the murine Clm-1-ECD-His fusion protein. Splenic B cells from the immunized animal were fused with myeloma to produce hybridomas. Positive clones were selected by ELISA, FACS, Western blot and immunohistochemical analysis based on reactivity with murine Clm-1. Clone 3F6 was selected for use in the study based on the criteria described above. Alexa fluorescent dye (488 or 647) -conjugated Clm-1 antibody Using AlexaProtein labeling kit (Invitrogen).

Activity Induction and clinical evaluation of EAE mice were treated with 200 μ g MOG in 200 μ l of emulsion containing 100 μ l PBS and 100 μ l Complete Freund's Adjuvant (CFA)_35-55Peptides were immunized subcutaneously. CFA was prepared by mixing incomplete Freund's adjuvant (DIFCO laboratory) with 8mg/ml M.tuberculosis (Mycobacterium tuberculosis) H37RA (non-viable and dry; DIFCO laboratory). Each mouse was also injected intraperitoneally with 200ng of pertussis toxin (Calbiochem) in 100 μ l pbs on day 0 and day 2 post immunization. Clinical signs were assessed using the following grading system: 0, no anomaly; 1, weak tail or hind limb weakness; 2, weak tail and hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, a moribund state. For the Clm-1-Fc fusion protein experiment, starting on day 0 of immunization, mice were treated subcutaneously three times weekly with 200 μ g of Clm-1-Fc fusion protein in 100 μ l PBS or with a control Fc protein (anti-gp 120). Data are reported as mean daily clinical score and standard error of the mean (SEM).

Bone marrow chimeras 6 week old C57BL/6(CD45.1) acceptorsMice were each lethally irradiated with two doses of 500 rad. Bone marrow cells from the femur and tibia were collected aseptically from C57BL/6(CD45.2) donor mice by flushing the bones with Hanks' balanced salt solution (HBSS; Hyclone) containing 5% FBS using a syringe and 27-gauge needle. Erythrocytes were lysed by ACK lysis buffer. Cells were washed in HBSS/FBS at 400g for 5 minutes, resuspended, and passed through a nylon mesh (BD Falcon) to remove debris. Then, the cells were washed twice with PBS and 10⁸Resuspend at individual cell/ml concentration. Irradiated recipient mice were injected 2x 10 via tail vein⁷Cells/200. mu.l. Reconstituted mice were kept in a pathogen-free facility for 8 weeks to allow for complete engraftment with donor bone marrow. Complete reconstitution of bone marrow was verified by FACS analysis of peripheral blood labeled for CD45.1 and CD45.2 isogenotypes in the lymphoid and myeloid compartments. EAE was induced in reconstituted recipient mice as described above.

Adoptive transfer of EAE As described for the induction of active EAE, Clm-1WT or KO mice were treated with MOG_35-55Peptide immunization except that mice were not injected with pertussis toxin. 10-12 days after immunization, draining (inguinal and brachial) lymph nodes were collected and triturated through a 70- μm cell strainer to obtain a single cell suspension. Cells were cultured in complete medium (RPMI 1640, 10% FBS, 2mM glutamine, 10mM HEPES, 1mM sodium pyruvate, 0.05mM beta-mercaptoethanol, 100U/ml penicillin, 100mg/ml streptomycin) at 5X10⁶The single cell/ml is treated with 20. mu.g/ml MOG_35-55Peptides and 20ng/ml recombinant murine IL-2 (R)&D system) was restimulated for 4 days. Recipient mice were injected via tail vein 10⁷And (4) cells. On the same day and two days later, recipient mice will also be injected with 200ng of pertussis toxin, as described above. Clinical assessment of EAE disease was performed as described above.

FACS analysis of spinal cord and lymph nodes when EAE disease is most severe (day 14-15 post immunization), mice were anesthetized and perfused with PBS containing 10U/ml heparin via the heart. The spinal cord was dissected and digested with collagenase D (2 mg/ml; Roche Diagnostics). By passing the tissue through a 70- μm cell strainer (BD bioscience), followed by a Percoll gradient (80% > -based-70%/60%/30%) to isolate monocytes. Cells were harvested from the 30%/60% interface and washed. Cells were also isolated from Draining Lymph Nodes (DLNs) as described above. Cells were Fc blocked with anti-Fc □ RIII/II for 30 min at 4 ℃ in FACS staining buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA). After washing, cells were stained with fluorescently conjugated mAbs for 30 min at 4 ℃. For intracellular staining of iNOS, cells were stained with antibodies against Clm-1(3F6), CD45, CD11b and CD11c, and then fixed with 3% paraformaldehyde in PBS solution at room temperature for 20 minutes. The cells were then resuspended in 100. mu.l of permeabilization solution (0.1% Triton-X in PBS). Cells were stained with 1 μ g/ml rabbit anti-iNOS antibody in permeabilization solution for 15 min at room temperature, followed by PE-labeled donkey anti-rabbit IgG for 15 min at room temperature. For analysis of Treg cells, single cell suspensions isolated from spinal cord and DLNs as described above were stained with CD45 and CD4, followed by intracellular staining of FoxP3 using the Cytofix/Cytoperm fixation/permeabilization kit (BD bioscience) according to the supplier's instructions. For intracellular staining of cytokines, cells were plated at 4x10 in 96-well round bottom plates⁵Individual cells/200. mu.l complete medium at 37 ℃ with 100. mu.g/ml MOG_35-55Peptide stimulation was carried out for 18-20 hours. Within the last 4 hours of stimulation, cells were treated with GolgiPlug (BD bioscience) at a 1: 1000 dilution. Intracellular staining for IL-17 and IFN □ was essentially performed as FoxP3 staining. Stained cells were analyzed by FACSCaliber or LSRII flow cytometer (Becton Dickinson). Data were analyzed using FlowJo software (Tree Star).

Expression of Clm-1 by Western blotting bone marrow-derived dendritic cells (BMDCs) were generated as described (Inaba et al, The Journal of Experimental Medicine 176, 16993-1702, 1992). In the presence of 10ng/ml GM-CSF (R)&D System (R)&DSystems)) were cultured and the medium was changed every three days. On day 7, cells were analyzed by FACS. BMDC purity of 90-95% CD11c⁺，CD11b⁺. Whole cell lysates from BMDCs were analyzed by immunoblotting with anti-Clm-1 Ab (3F6) using standard methods.

Clm-1 expression real-time PCR analysis spinal cords and DLNs were isolated from mice on days 0, 7, 14 and 21 of EAE as described above. Total RNA was isolated using RNeasy protection Mini Kit (RNeasy ProtectMini Kit) (QIAGEN). cDNA was synthesized using 1. mu.g of gRNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Clm-1mRNA and 18s rRNA were measured using TaqMan Universal PCRMaster mix and validation primer and probe sets (Mm00467508_ m1 and Hs03003631_ g1) (Applied Biosystems), respectively.

Measurements of cytokine and nitric oxide production monocytes were isolated from spinal cord on day 15 of EAE as described above. Single cell suspension in 96-well round bottom plates at 37 ℃ in the absence or presence of 100. mu.g/ml MOG_35-55Complete medium of peptide (5X 10)⁵Cells/200. mu.l). Culture supernatants were collected after 36 hours. Cytokine release was measured by Luminex using the Bio-Plex mouse cytokine 23-Plex panel (Bio-Rad). Nitric oxide production was measured using the Griess assay (Promega) according to the supplier's instructions.

In vitro antigen-specific recall reactions as described above, draining lymph nodes were collected from mice on day 14 of EAE. Single cell suspension in 96-well round bottom plates at 37 ℃ with or without titrating MOG_35-55Complete medium of peptide (5X 10)⁵Individual cells/200 μ l) for 3 days. Then, the cells were treated with 0.5. mu. Ci/well [ 2 ]³H]The last 6 hours of thymidine pulse incubation. Detected by using a Topcount Microplate liquid Scintillation Counter (Topcount Microplate science Counter) (Packard instruments)³H]Thymidine uptake determines proliferation. Alternatively, supernatants were collected on day 3 for cytokine analysis. Cytokine measurements were performed by ELISA (BD Biosciences).

Immunohistochemistry on the indicated days post-immunization, mice were anesthetized as described above and perfused with 30ml of PBS followed by 10ml of 4% Paraformaldehyde (PFA). The spinal cord was removed by dissection and fixed in 4% PFA overnight, then submerged in 10%, 20%, 40% sucrose in sequenceIn a sugar solution. The spinal cords were then frozen in OCT on dry ice and stored at-80 ℃ in plastic bags to prevent dehydration. Seven micron thick cross-sections were cut and mounted on Superfrost Plus slides (Fisher Scientific). For Clm-1 and CD45.2 co-staining, slides were blocked with hamster serum and biotin blocking kit (Sigma). Tissues were stained with hamster anti-Clm-1 (3F6) and biotin-conjugated anti-CD 45.2 and then detected with Cy 3-anti-hamster IgG and Alexa Fluor 488-streptavidin (Invitrogen). For Clm-1 and CD11c co-staining, slides were first stained with anti-Clm-1 and then detected with Cy 3-anti-hamster IgG. The slides were then stained with biotin-anti-CD 11c (HL3) and detected with Alexa Fluor 488-streptavidin. For the co-staining of myelin and CD11c, slides were first stained with biotin-anti-CD 11c and detected with Alexa Fluor 594-streptavidin (Invitrogen). Then, the myelin is applied to FluoroMyelin^TMStaining with green fluorescent myelin staining kit (Invitrogen). Sections were coverslipped with extended Gold anti-fade media (Invitrogen) with DAPI. Slides were examined and pictures were captured using an Olympus BX61 fluorescence microscope. To detect the extent of demyelination, cervical and thoracic spinal cord sections were sectioned with FluoroMyelin^TMAnd (3) dyeing by using a green fluorescent myelin dyeing kit. The demyelinated area was estimated by manually tracing the total cross-sectional area and the demyelinated area of each section. Total demyelination is expressed as a percentage of total spinal cord area.

Statistical analysis comparisons of EAE clinical scores, demyelination or other cell counts and cytokine production between any two groups of mice were made by a two-tailed paired student's t-test, assuming unequal variances. p values < 0.05 were considered significant.

Results and discussion

At the site of CNS inflammation, Clm-1 is expressed on TNF-and iNOD-producing CD11c + cells

Clm-1 was first searched for a gene encoding a single transmembrane, containing immunityBioinformatic pathway identification of genomic predicted sequences of members of the Ig-superfamily of tyrosine inhibitory motifs (ITIM) (Abbas et al, Genes and Immunity 6, 319-331, 2005). Candidate mouse homologues of ITIM-containing genes were then selected based on changes in expression levels in the spinal cord following immunization with Myelin Oligodendrocyte Glycoprotein (MOG) peptide. And for the first time used in the experiment () Clm-1 expression increased more than 100-fold when disease was most severe compared to mice (FIG. 1A, left panel). Monoclonal antibodies directed against the extracellular domain of CLM-1 were generated to determine the cellular origin of CLM-1. In naive mice, CLM-1 was absent on the local microglia population (fig. 1B). In spinal cords from MOG-immunized mice, CLM-1 was expressed on CD11b/CD11C double positive cells with high MHC class II and CD86 expression (fig. 1C). At the onset of disease, CLM-1CD11c double positive cells were distributed along the medulla membrane and blood vessels (results not shown). In the most severe cases, Clm-1+ cells were clustered in the white matter of the dorsal and ventral horns of the thoracic and lumbar spinal cords (FIG. 1D). Further analysis showed that Clm-1+ cells expressed iNOS and TNF (FIG. 1E), and were therefore phenotypically similar to Tip-DCs, which were originally described as a subset of bone marrow cells required for efficient pathogen elimination (Serbina et al, Immunity 19, 59-70, 2003). TipDCs and their precursors have been identified in subsequent EAE studies as pathogenic effector cells that contribute to the pathogenesis of EAE (King et al, Blood 113, 3190-3197, 2009). Thus, increased expression of CLM-1 on inflammatory bone marrow cells in the CNS may indicate a regulatory function in the pathogenesis of EAE.

CLM-1 is expressed on circulating Ly6+ bone marrow precursors migrating to the CNS during autoimmune demyelinating diseases

To further determine the myeloid lineage from which CLM-1 positive cells are derived, we utilized a Cx3cr1+/gfp reporter strain, which expresses green fluorescent protein in cells of the monocyte and macrophage/dendritic cell lineages (Geissmann et al, Immunity 19, 71-82,2003). In peripheral blood, after MOG immunization, CLM-1 was at Cx3cr1^loLy6C^hiCD115⁺CD62L⁺Ly6G^-Inflammatory monocytes, but not Cx3cr1 in naive and immunized mice^hiCD11c⁺On The normal DC precursor (FIG. 2A) (Auffray et al, The Journal of Experimental Medicine 206, 595-. To further determine whether CLM-1 positive cells in the inflamed CNS are indeed derived from radiation-sensitive bone marrow-derived cells and not from radiation-resistant CNS microglia, mice with a CD45.1 allotype were irradiated and reconstituted with donor cells with a CD45.2 allotype. CLM-1 expression was not present on irradiation-resistant microglia, but on bone marrow-derived donor cells that home to the CNS (fig. 2B). These results were confirmed, and CLM-1 was not present in Cxcr3 of spinal cord used for the first experiment^hiColonize microglia, but in the most severe cases, Cx3cr1⁺CD11c⁺High expression on subpopulations of cells (fig. 2C). Discovery of CLM-1⁺Cx3cr1^loDouble positive cells were adjacent to the medullary membrane of the thoracic and lumbar spinal cords and the dorsal and ventral horns of the median eminence, but remained absent on resident microglia located in the gray matter of the dorsal and ventral horns of the spinal cord (fig. 2D). Taken together, these results indicate that CLM-1 is expressed on inflammatory monocytes and bone marrow-derived DCs in CNS inflammatory lesions, but not on circulating common DC precursors or CNS-resident microglia.

Lack of CLM-1 leads to increased disease severity of MOG-induced EAE

Since CLM-1 contains two ITIMs and one ITSM motif in its cytoplasmic domain (Chung et al, J Immunol 171, 6541-6548, 2003) and is able to recruit SHP-1 after cross-linking with activating receptors in a forced overexpression system (Izawa et al, The Journal of biological Chemistry 282, 17997-18008, 2007), we determined whether CLM-1 can act to inhibit inflammatory responses in MOG-induced EAE. By homologous recombination, mice lacking CLM-1 exon 1 were generated, which resulted in a deficiency of transcripts and proteins (supplementary figure 1). CLM-1ko mice were viable and born at the predicted mendelian ratio. Mice did not differ in body weight or skeletal parameters when measured at 6,9 and 12 weeks of age (results not shown). Bone marrow and lymphocyte subsets in the inguinal lymph node, spleen and blood were similar in CLM-1ko and wt mice (results not shown). Successful elimination of the CLM-1 gene in ko mice was verified by flow cytometry and Western blot analysis (fig. 3A, left panel). On bone marrow derived dcs (bmdcs) from CLM-1wt and ko mice, the expression levels of cell surface molecules associated with antigen presentation and co-stimulation were similar (fig. 3A, right panel), as were the expression levels of other members of the CMRF cluster (results not shown). In CLM-1wt and ko mice, the dendritic cell morphology (fig. 3B, left panel) and number (fig. 3B, right panel) of the various inflammatory cell populations were similar when the disease was most severe. When MOG was immunized, both CLM-1wt and ko mice developed disease at similar incidence. However, in mice lacking CLM-1, the severity of the disease increased significantly (fig. 3C). To determine whether this phenotype was due to binding of putative ligands to CLM-1, mice were treated with CLM-1 soluble counterparts (CLM-1-Fc fusion proteins). Consistent with the results obtained in CLM-1ko mice, disease severity was significantly increased in CLM-1-Fc treated mice, while disease incidence remained similar, compared to mice treated with the control fusion protein (fig. 3D). Thus, lack of CLM-1 receptor function leads to increased disease severity, indicating a potential inhibitory effect in CNS inflammation.

Clm-1 do not regulate T cell priming

A splice variant of CLM-1, Digr1, was previously identified as a negative regulator of T cell responses (Shi et al, Blood 108, 2678-2686, 2006). Since EAE can be induced by antigen-specific T cell priming, we further determined whether CLM-1 affects T cell responses. Spleen cDCs or BMDCs derived from CLM-1wt and ko mice were incubated with allogeneic T cells or with T cells expressing TCR specific for OVA peptides. Proliferation (FIG. 2 supplemented) and cytokine response (results not shown) were independent of CLM-1 status. To further determine whether CLM-1 affected T cell sensitization in vivo, T cells isolated from peripheral lymph nodes 7 days after MOG immunization were isolated and re-stimulated with MOG peptide. CLM-1 status did not affect T cell proliferation, cytokine response or production of Foxp3 regulatory T cells in Peripheral Lymph Node (PLN) cells (fig. 4A and supplementary fig. 3 a). Finally, to consolidate the role of CLM-1 in modulating T cell effector function in vivo, T cells from CLM-1wt and ko donors were adoptively transferred to ko and wt acceptors, respectively. Disease severity was not affected by CLM-1 status in T cell donors, but was significantly increased in T cell recipients lacking CLM-1 (fig. 4B). This suggests that CLM-1 functions to modulate disease severity during the effector phase, rather than during the initial T cell priming phase, following MOG immunization.

Next, we verified whether CLM-1 affected the reactivation of CNS-infiltrating CD4+ T cells and the cytotoxic activity of inflammatory DCs. CNS leukocytes harvested from the spinal cord when the disease was most severe and re-stimulated with MOG peptides in the presence of antigen presenting cells showed similar polarization and similar T cell-specific cytokine responses against Th1, Th17 and Foxp3Treg cells (fig. 5A and supplementary fig. 3 b). In contrast, leukocytes obtained from spinal cords of CLM-1ko mice produced significantly elevated levels of nitric oxide and bone marrow-specific proinflammatory cytokines compared to wt mice (fig. 5B). Thus, in MOG-induced EAE, CLM-1 down-regulates bone marrow effector function without affecting T cell responses.

We next determined whether increased paralysis in CLM-1ko mice immunized with MOG peptides was caused by increased activity of bone marrow cells in the spinal cord. CLM-1 positive cells were found to cluster at demyelinating sites in the dorsal horn of the cervical and thoracic spinal cords. Cells were found to be opposite and generally surrounding myelin folds (fig. 6A), and in some cases MOG-positive myelin residues were present within CLM-1 positive cells (results not shown). Since CLM-1 is an inhibitory receptor and the extent of infiltrating myeloid cells is similar in CLM-1wt and ko mice, we theorize that the lack of CLM-1 may result in increased activation and effector activity per cell, which leads to increased demyelination. The absence of CLM-1 resulted in increased demyelination (fig. 6B and C), indicating increased cytotoxic activity in CD11C + cells lacking CLM-1. Thus, CLM-1 down-regulates bone marrow cell activation, which produces an interruption in axonal demyelination in the spinal cord.

Despite the steadily increasing number of receptors containing ITIM sequences in their intracellular domains, little is known about the biological role of many of these receptors. This study identified CLM-1 for the first time as an inhibitory receptor on CNS infiltrating inflammatory DCs. We further show that soluble counterparts of this receptor can exacerbate disease severity, suggesting that the extracellular domain acts as a decoy receptor for certain yet unidentified ligands. Although the identification of putative ligands would undoubtedly increase our understanding of CLM-1 biology, this study clearly demonstrates that CLM-1 plays a non-redundant role in controlling myeloid cell activation and demyelination in the CNS.

Claims (26)

1. A method for treating a demyelinating disease in a mammalian subject, the method comprising administering to the subject an effective amount of a CLM-1 agonist.

2. The method of claim 1, wherein the mammalian subject is a human.

3. The method of claim 2 wherein the demyelinating disease is a demyelinating autoimmune disease.

4. The method of claim 3 wherein the demyelinating autoimmune disease affects the Central Nervous System (CNS).

5. The method of claim 4 wherein the demyelinating autoimmune disease is selected from the group consisting of: multiple Sclerosis (MS), relapsing remitting MS (rrms), primary and secondary progressive forms of MS, progressive relapsing forms of MS, encephalomyelitis, leukoencephalitis, transverse myelitis, neuromyelitis optica (devike's disease), and optic neuritis.

6. The method of claim 5 wherein the demyelinating autoimmune disease is MS.

7. The method of claim 3 wherein the demyelinating autoimmune disease affects the peripheral nervous system.

8. The method of claim 7 wherein the demyelinating autoimmune disease is selected from the group consisting of: acute inflammatory demyelinating polyneuropathy (AIDP; Guillain-Barre syndrome); chronic inflammatory demyelinating polyneuropathy; anti-MAG peripheral neuropathy; and Motor and Sensory Neuropathy (HMSN) (also known as Hereditary Sensory Motor Neuropathy (HSMN), or peroneal muscular atrophy, or progressive neuropathic peroneal muscular atrophy).

9. The method of any one of claims 1-8 wherein the CLM-1 agonist is an agonist anti-CLM-1 antibody.

10. A pharmaceutical composition for the treatment of demyelinating diseases comprising an effective amount of a CLM-1 agonist in admixture with a pharmaceutically acceptable excipient.

11. The pharmaceutical composition of claim 11, wherein the demyelinating disease is a demyelinating autoimmune disease.

12. The pharmaceutical composition of claim 11, wherein the demyelinating autoimmune disease is Multiple Sclerosis (MS).

13. Use of an effective amount of a CLM-1 agonist in the manufacture of a medicament for the treatment of a demyelinating disease.

14. The use of claim 13 wherein the demyelinating disease is a demyelinating autoimmune disease.

15. The use of claim 11, wherein 14 the demyelinating autoimmune disease is Multiple Sclerosis (MS).

16. The use of any one of claims 13 to 15 wherein the CLM-1 agonist is an agonist anti-CLM-1 antibody.

17. CLM-1 agonists for the treatment of demyelinating diseases.

18. The CLM-1 agonist of claim 17 wherein the demyelinating disease is a demyelinating autoimmune disease.

19. The CLM-1 agonist of claim 18 wherein the demyelinating autoimmune disease is MS.

20. The CLM-1 agonist of any one of claims 17 to 19 wherein the CLM-1 agonist is an agonist anti-CLM-1 antibody.

21. A method for diagnosing a demyelinating disease, the method comprising detecting a defect in CLM-1 function.

22. The method of claim 21 wherein the demyelinating disease is a demyelinating autoimmune disease.

23. The method of claim 22 wherein the demyelinating autoimmune disease is MS.

24. A kit for treating a demyelinating disease comprising a CLM-1 agonist and instructions for use.

25. The kit of claim 24 wherein the demyelinating disease is a demyelinating autoimmune disease.

26. The kit of claim 25 wherein the demyelinating autoimmune disease is MS.