WO2007101254A2 - Compositions and methods for the treatment of immune system disorders - Google Patents

Abstract

The invention provides methods for ameliorating a sign or symptom associated with an autoimmune disease by administering a dimeric TIM-I ligand targeting molecule to a subject. The invention additionally provides methods for ameliorating a sign or symptom associated with transplant rejection by administering a dimeric TIM-I ligand targeting molecule to a subject.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF IMMUNE SYSTEM DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application number

60/777,958 filed on February 28, 2006. The aforementioned application is herein incorporated by this reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] This invention relates generally to the area of immunology and more specifically to treating immune system disorders.

[0003] Recently, a novel family of protein molecules called TIMs (T cell

Immunoglobulin and Mucin domain containing proteins) has been identified whose members appear to exert great influence over the type of immune response (ThI or Th2) that develops following immunization (Kuchroo et al., Nat. Rev. Immunol. 3:454-462 (2003); Monney et al., Nature 415:536-541 (2002)). The TIM family consists of eight members in mice (TIM-I through TIM-8), and three proteins in human, TIMs-I, -3, and - 4, which are homologous to mouse TIM-l/TIM-2, TIM-3, and TIM-4, respectively (Kuchroo et al., supra, 2003). These proteins are similar in structure, consisting of a characteristic immunoglobulin V (IgV) domain, mucin-like domain, transmembrane helix, and cytoplasmic domain (Kuchroo et al., supra, 2003).

[0004] While a role for the TIMs, and in particular for TIM-I, in regulating susceptibility to atopic diseases is well established (Chae et al., Hum. Immunol. 64:1177- 1182 (2003); Chae et al.. Biochem. Biophys. Res. Commun. 312:346-350 (2003); Chae et al., Biochem. Biophys. Res. Commun. 315:971-975 (2004); Gao et al., J. Allergy Clin.Immunol. 115:982-988 (2005); Mclntire et al., Nature 425:576 (2003), more recent data suggest broad immunoregulatory functions for the various TIM family members. For instance, molecules targeting the TIM-signaling pathways, such as TIM-specific monoclonal antibodies and recombinant TIM proteins, have been shown to greatly affect immune responses to antigen challenge. Studies treating mice with either TIM-3 blocking antibody or TIM-3/Fc fusion proteins suggest that the natural interaction of TIM-3 with its cognate ligand may normally downregulate ThI responses in vivo, possibly playing an important role in the maintenance of peripheral tolerance (Monney et al., Nature 415:536- 541 (2002); Sabatos et al., Nat. Immunol. 4: 1102-1110 (2003); Sanchez-Fueyo et al., Nat. Immunol. 4:1093-1101 (2003)).

[0005] Characterization of TIM-I biological activity has also yielded intriguing results. However, published studies are not conclusive. While stimulation of BALB/c CD4+ T cells with a TIM-I -specific monoclonal antibody plus TCR and CD28 signaling significantly enhanced T cell proliferation and production of Th2 cytokines in vitro, the same antibody elicited a more mixed response in vivo, driving CD4+ T cell proliferation and IL-4 secretion, together with vigorous production of IFN-gamma (Umetsu et al., Nat. Immunol. 6:447-454 (2005)). TIM-4 has been described as a natural ligand for TIM-I, with expression restricted to macrophages and mature dendritic cells (Meyers et al., Nat. Immunol. 6:455-464 (2005)). Experiments providing TIM-4/Fc fusion protein in vivo revealed that signaling through the TIM-I pathway induces T cell hyperproliferation ex vivo, with robust production of IFN-gamma and IL-2, but not IL-4 and only minimal IL- 10 (Meyers et al., supra, 2005)).

[0006] Since TIM molecules and signaling pathways influence the type of immune response, in particular affecting ThI and Th2 responses, agents that modulate TIM signaling can be used to modulate an immune response. Thus, it is desirable to identify agents that modulate TIM signaling. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0007] The invention provides methods for ameliorating a sign or symptom associated with an autoimmune disease by administering a dimeric TIM-I ligand targeting molecule to a subject. The invention additionally provides methods for ameliorating a sign or symptom associated with transplant rejection by administering a dimeric TIM-I ligand targeting molecule to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows a schematic map of a TIM-I /Fc expression vector plasmid. [0009] Figure 2 shows the experimental design for the induction of experimental autoimmune encephalitis (EAE) and administration schedule for ML- mTIM-lFc- and control IgG2a Mab.

[0010] Figure 3 shows the incidence of EAE in mice (n = 22) treated with ML- mTIM-1/Fc compared with those treated with control IgG2a antibody (n = 26). The incidence in the control group was significantly higher than that observed in the ML- mTIM-1/Fc treated group (p < 0.02). Data were obtained from 3 independent experiments.

[0011] Figure 4 shows a dot plot for the day of onset of EAE. Each dot represents a single mouse. The days of onset of EAE for the mice (n=9/22) treated with ML-mTIM- 1/Fc were compared with those treated with control IgG2a antibody (n=25/26). Both groups were compared using the Mann- Whitney unpaired t-test. The difference was statistically significant (p < 0.006). Data were obtained from 3 independent experiments.

[0012] Figure 5 shows a dot plot for the maximum disease score observed. Each dot represents a single mouse. The maximum disease score of the mice (n=22) treated with ML-mTIM-1/Fc was compared with those treated with control IgG2a antibody (n=26). Both groups were compared using the Mann- Whitney unpaired t-test. The difference was statistically significant (p < 0.0001). Data were obtained from 3 independent experiments.

[0013] Figure 6 shows that treatment with TIM-l/Fc prevents disease development in the mouse model of EAE. The mean disease score of the mice treated with ML-mTIM- 1/Fc (n=22) versus the mice treated with control IgG2a antibody (n=26) over time is shown.

[0014] Figure 7 shows the leader sequence of human CD5 (nucleotide sequence,

SEQ ID NO: 1; amino acid sequence, SEQ ID NO:2).

[0015] Figure 8 shows nucleotide and amino acid sequences of the extracellular domains of the mouse the TIM-I Balb/c allele: Ig domain nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences; Ig+mucin domain nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO: 6) sequences. [0016] Figure 9 shows nucleotide and amino acid sequences of the extracellular domains of the mouse TIM-I C57BL6 allele: Ig domain nucleotide (SEQ ID NO:7) and amino acid (SEQ ID NO:8) sequences; Ig+mucin nucleotide (SEQ ID NO:9) and amino acid (SEQ ID NO: 10) sequences.

[0017] Figure 10 shows nucleotide and amino acid sequences of the extracellular domains of human TIM-I : Ig domain nucleotide (SEQ ID NO: 11) and amino acid (SEQ ID NO: 12) sequences; Ig+mucin domain nucleotide (SEQ ID NO: 13) and amino acid (SEQ ID NO: 14) sequences.

[0018] Figures 1 IA-I IE show the nucleotide and amino acid sequences of various human TIM-I alleles. Figure HA shows the nucleotide (SEQ ID NO: 15) and amino acid (SEQ ID NO: 16) sequences for human TIM-I allele 1. The human TIM-I allele set forth in Figure 1 IA contains an insertion (MTTTVP) after position 157 in the human TIM-I amino acid sequence. Figure 1 IB shows the nucleotide (SEQ ID NO: 17) and amino acid (SEQ ID NO: 18) sequences for human TIM-I allele 2. Figure 11C shows the nucleotide (SEQ ID NO: 19) and amino acid (SEQ ID NO:20) sequences for human TIM-I allele 3. Figure HD shows the nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences for human TIM-I allele 4. Figure 1 IE shows the nucleotide (SEQ ID NO:23) and amino acid (SEQ ID NO:24) sequences for human TIM-I allele 5; the nucleotide (SEQ ID NO:25) and amino acid (SEQ ID NO:26) sequences for human TIM-I allele 6; the nucleotide (SEQ ID NO:27) and amino acid (SEQ ID NO:28) sequences for human TIM-I allele 7; the nucleotide (SEQ ID NO:29) and amino acid (SEQ ID NO:30) sequences for human TIM-I allele 8; the nucleotide (SEQ ID NO:31) and amino acid (SEQ ID NO:32) sequences for human TIM-I allele 9; the nucleotide (SEQ ID NO:33) and amino acid (SEQ ID NO:34) sequences for human TIM-I allele 10; the nucleotide (SEQ ID NO:35) and amino acid (SEQ ID NO:36) sequences for human TIM-I allele 11. It is to be understood that the present invention also provides the amino acid sequences of TIM-I alleles comprising any combination of the insertions, substitutions, and deletions set forth in Figures 1 IA-I IE. Nucleic acid sequences encoding these alleles are also provided. For example, and not to be limiting, an amino acid sequence for a TIM-I allele can comprise one or more of the substitutions set forth in Figure 1 IE. A TIM-I allele can also comprise one or more of the substitutions set forth in Figure 11 E with or without an insertion (MTTTVP) after position 157 in the human TIM-I amino acid sequence. [0019] Figure 12 shows nucleotide and amino acid sequences of mouse IgG2aFc

(hinge, CH2 and CH3 domains): lytic nucleotide (SEQ ID NO: 37) and amino acid (SEQ ID NO:38) sequences; non-lytic nucleotide (SEQ ID NO:39) and amino acid (SEQ ID NO:40) sequences.

[0020] Figure 13 shows nucleotide and amino acid sequences of human IgGlFc:

IgGl hinge + CH2 + CH3 domain nucleotide (SEQ ID NO:41) and amino acid (SEQ ID NO:42) sequences.

[0021] Figure 14 shows nucleotide and amino acid sequences of human IgG4Fc:

IgG4 hinge + CH2 + CH3 domain nucleotide (SEQ ID NO:43) and amino acid (SEQ ID NO:44) sequences.

[0022] Figure 15 shows an exemplary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot of purified full length and mucinless TIM- 1/Fc.

[0023] Figure 16 shows the experimental protocol for treatment of alopecia areata with TIM-I /Fc.

[0024] Figures 17A and 17B show that TIM-I/ Fc reduces hair loss in recipient

C3H/ HeJ mice. Mice who received TIM-I/ Fc had a significantly smaller area of alopecia compared to control mice. Figure 17 A shows a graph of affected surface area (p=<0.05). Figure 17B shows photographs of four representative treated and control animals.

[0025] Figure 18 shows lymphocyte proliferation in a C3H/HeJ mouse model of alopecia areata. TIM-I /Fc (n=4) treated mice display stronger in vitro Con A stimulated lymphocyte proliferation compared to control animals (n=4) (P<0.05).

[0026] Figures 19A-19E show that TIM-I /Fc treated mice (n=4) displayed increased (P<0.05) production of Th2 cytokines in vitro after Con A stimulation compared to controls (n=4) for IL-4 (Figure 19A) and IL-9 (Figure 19C), with trends of increased IL-5 (Figure 19B), IL-10(Figure 19D), and IL- 13 (Figure 19E). [0027] Figure 20 shows the experimental design for the induction of experimental autoimmune encephalitis (EAE) and administration schedule for anti-TIM-4 monoclonal antibodies and control IgG2a Mab.

[0028] Figure 21 shows the incidence of EAE in mice (n = 6) treated with anti-

TIM-4 compared with those treated with control IgG2a antibody (n = 6) over time. The incidence in the control group was significantly higher than that observed in the anti- TIM-4 treated group.

[0029] Figure 22 shows that treatment with anti-TIM-4 prevents disease development in the mouse model of EAE. The mean disease score of the mice treated with anti-TIM-4 (n=6) versus the mice treated with control IgG2a antibody (n=6) over time is shown.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The invention provides a novel agent for the treatment of immunological disorders, in particular, autoimmune diseases and transplant rejection. The novel agent is a dimeric molecule that specifically interacts with TIM-I ligands. Such agents of the invention include, for example, dimeric TIM-I proteins. Such agents additionally include TIM-4 antibodies, TIM-4 being a specific TIM-I ligand. Dimeric TIM-I proteins comprise, for example, TIM-I proteins, which are dimerized (a) through the Fc domain of an antibody molecule, (b) through an amino acid linker domain, (c) through a chemical cross-linker, (d) through crosslinking to a carrier protein, such as BSA, or (e) through crosslinking by covalent or non-covalent attachment to a solid surface, for example, microcarrier beads or the like. The TIM-I portion of the dimeric agent is the extracellular Ig domain of TIM-I and does not contain the extracellular mucin domain of the protein. Anti-TIM-4 antibodies comprise dimeric TIM-4 antibodies, which are capable of dimerizing the TIM-I ligand TIM-4. These include full length dimeric TIM-4 antibodies of an IgG isotype, but also TIM-4 antibody fragments which contain the antigen-binding determinants of the antibodies and which are dimerized as above for TIM-I /Fc.

[0031] The agents of the invention, and specifically a TIM-I /Fc fusion protein and

TIM-4 antibodies, have been found to prevent disease development in an animal model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE) model, and in an animal model of alopecia areata (see Examples). This finding has not been previously described and is unexpected, as TIM-I has previously been described to induce T cell activation and stimulate immune responses, rather than inhibiting immune responses as disclosed herein as effective for treating an autoimmune disease or other condition that involves pathogenic T cell responses, such as transplant rejection (Umetsu et al., supra, 2005). Thus, the compositions and methods of the invention are based on the use of dimeric TIM-I ligand targeting agents for the treatment of immunological disorders, and in particular for the treatment of autoimmune diseases and transplant rejection.

[0032] As disclosed herein, a recombinant, dimeric TIM-I protein has been generated. Both mouse and human TIM-I dimeric proteins have been generated (see Examples). An exemplary dimeric TIM-I protein consists of the extracellular Ig domain of TIM-I fused to the hinge, CH2 and CH3 domains of an antibody (IgG) Fc domain. Alternatively, a dimeric TIM-I protein consists of the complete extracellular domain of TIM-I (Ig and mucin domains) fused to an Ig Fc domain. Dimerization of the molecule is mediated through an interchain disulfide bond between the two respective Fc domains of the resulting dimeric molecule. In addition, mouse and human anti-TIM-4 monoclonal antibodies have been generated.

[0033] As disclosed herein, both a recombinant, dimeric TIM-I protein containing the extracellular Ig domain of TIM-I and a monoclonal antibody against TIM-4 are capable of preventing disease development in a mouse model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE) model (Example IV). A recombinant dimeric TIM-I protein is also capable of preventing disease development and disease progression in a mouse model of alopecia areata (see Example V).

[0034] In one embodiment, the invention provides a method for ameliorating a sign or symptom associated with an autoimmune disease by administering a TIM-I ligand targeting molecule to a subject, wherein the TIM-I ligand targeting molecule is dimeric. The TIM-I ligand targeting molecule can be, for example, a TIM-4 antibody. The TIM-I ligand targeting molecule can also be a TIM-I-Fc fusion polypeptide. The Fc portion of a TIM-I-Fc fusion polypeptide or of a TIM-4 antibody can be target-cell depleting or non target-cell depleting. In one particular embodiment, the TIM-I ligand targeting molecule is a TIM-I-Fc fusion polypeptide containing a TIM-I Ig domain. For example, the TIM- 1-Fc fusion polypeptide can contain a TIM-I Ig domain in the absence of a TIM-I mucin domain, also referred to herein as mucinless. [0035] In a particular embodiment, the invention provides a method for ameliorating a sign or symptom associated with multiple sclerosis by administering a TIM-I ligand targeting molecule to a subject, wherein the TIM-I ligand targeting molecule is dimeric. Multiple sclerosis (MS) is considered to be a prototype ThI mediated inflammatory autoimmune disorder of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating syndrome induced in rodents by active immunization with myelin specific components, for example, myelin basic proteins (MBP) and proteolipid proteins (PLP). Several rodent models for EAE have been established. The SJL/J mouse model presents the disease in a relapsing-remitting manner that is similar to multiple sclerosis (MS) in humans. It is therefore widely used to evaluate the therapeutic efficacy of new drug candidates for MS. Various immunomodulatory approaches used to currently treat MS include IFN-β, gatiramer acetate, cyclophosphamide, corticosteroids, cannabinoids and mitoxanthrone. The anti VLA-4 monoclonal antibody that had recently shown promise has been withdrawn due to fatal effects in a fraction of patients. Although the cell-replacement strategies in animal models yield promising results, translating these findings into an effective therapy has a long way to go. In spite of the several therapeutic approaches currently available, options for fighting MS thus remain limited. As disclosed herein, a dimerized form of mouse mucinless TIM-I /Fc fusion protein and TIM-4 antibody lowers the incidence of disease, delays the onset of disease, and suppresses the severity of disease in a disease model of relapsing-remitting EAE in SJL/J mice (Example IV and Example VI).

[0036] In another embodiment of a method of ameliorating a sign or symptom associated with an autoimmune disease, the autoimmune disease can be selected from rheumatoid arthritis, autoimmune diabetes mellitus, systemic lupus erythematosus, autoimmune lymphoproliferative syndrome (ALPS), alopecia areata and inflammatory bowel disease. Other exemplary autoimmune diseases that can be treated by a method of the invention using a dimeric TIM-ligand targeting molecule include, for example, rheumatoid arthritis, autoimmune diabetes mellitus, systemic lupus erythematosus, psoriasis, psoriatic arthritis, an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis, myasthenia gravis and autoimmune lymphoproliferative syndrome (ALPS), as well as atherosclerosis and Alzheimer's disease, or other autoimmune diseases, as disclosed herein. Autoimmune disorders are mediated by cellular effectors, for example, T cells, macrophages, B cells and the antibodies they produce, and others cells. These cells express one or more TIM or TIM ligands, as disclosed herein. By eliminating the cells involved in an autoimmune response, for example, using a lytic Fc in an antibody or fusion protein, or by using a toxic conjugate, a therapeutic benefit is achieved in such an autoimmune disorder. These and other autoimmune diseases, as disclosed herein, can be treated by a method of the invention to ameliorate a sign or symptom associated with a particular autoimmune disease. One skilled in the art can readily determine appropriate signs or symptoms associated with a particular disease and recognize whether such signs or symptoms are ameliorated.

[0037] The invention provides agents that target the TIM signaling pathways and particularly the TIM-I and TIM-4 signaling pathways. As used herein, the term "agent," when used in reference to a TIM signaling pathway, refers to a molecule that modulates a signaling pathway mediated by a TIM, in particular TIM-I or TIM-4. A TIM targeting agent is also referred to herein as a TIM targeting molecule or reagent. As used herein, a "TIM-I ligand targeting molecule" or agent is a molecule or agent that specifically binds to a TIM-I ligand, particularly in dimeric form. Such agents include fragments of TIM-I that binds to a TIM-I ligand or antibodies against TIM-I ligands, in particular antibodies against TIM-4, a TIM-I ligand. TIM-I ligand binding fragments of TIM-I include TIM-I fusion proteins. Fusion proteins include, for example, fusions of TIM-I or TIM-I ligands with proteins or protein fragments, such as with the Fc region of immunoglobulins, with albumin, with transferrin, with a Myc tag, with a polyhistidine tag or other desired proteins or protein fragments. Agents of the invention also include chemically modified agents, such as pegylated TIM or TIM ligands or other desired chemical modifications. Agents of the invention also include agents attached to a solid support, for example, TIM or TIM ligands attached to microcarrier beads or other solid supports. It is understood that, when referring to a particular TIM, polymorphic and splice variants of that TIM are included. An agent of the invention can also be a small molecule, a peptide, a polypeptide, a polynucleotide, including antisense and siRNAs, a carbohydrate including a polysaccharide, a lipid, a drug, as well as mimetics, derivatives and combinations thereof that stimulate or inhibit interaction of a specific TIM, in particular TIM-I, with its ligands, or stimulate or inhibit TIM-I or TIM-I ligand signaling. [0038] As used herein, a "TIM targeting molecule" refers to a molecule that binds to a TIM or TIM ligand. Exemplary TIM targeting molecules include, but are not limited to, antibodies against a TIM, antibodies against a TIM ligand, a recombinant TIM protein, a TIM fusion polypeptide, a TIM ligand, including a TIM ligand fusion polypeptide. As disclosed herein, an antigen and TIM targeting molecule or agent can be administered in a single composition or as separate compositions.

[0039] Various TIMs are well known to those skilled in the art, including TIM-I,

TIM-2, TIM-3 and TIM-4. Various TIMs are taught, for example, in WO 03/002722; WO 97/44460; U.S. Patent No. 5,622,861, issued April 22, 1997; U.S. Patent No. 6,664,385, issued December 16, 2003; U.S. publication 2003/0124114; Bailly et al., L Biol. Chem. 277:39739-39748 (2002), each of which is incorporated herein by reference. Exemplary TIM sequences are shown in Figures 8-11. A variety of TIMs from different species can be used in compositions and methods of the invention, depending on the desired use. A TIM from a particular species can be used for a particular use, for example, a human TIM can be used in a human, if desired. TIMs from other species can also be used, as desired.

[0040] In one embodiment, a TIM-I ligand targeting molecule can be, for example, a fusion protein with TIM-I and can include at least one domain or portion thereof of an extracellular region of TIM-I and a constant heavy chain or portion thereof of an immunoglobulin. In a particular embodiment, a soluble TIM fusion protein refers to a fusion protein that includes at least one domain of an extracellular domain of a TIM and another polypeptide. In one embodiment, the soluble TIM can be a fusion protein including the extracellular region of a TIM covalently linked, for example, via a peptide bond, to an Fc fragment of an immunoglobulin such as IgG; such a fusion protein typically is a homodimer. In another embodiment, the soluble TIM fusion can be a fusion protein including just the Ig domain of the extracellular region of a TIM covalently linked, for example, via a peptide bond, to an Fc fragment of an immunoglobulin such as IgG; such a fusion protein typically is a homodimer. As is well known in the art, an Fc fragment is a homodimer of two partial constant heavy chains. Each constant heavy chain includes at least a CHl domain, the hinge, and CH2 and CH3 domains. Each monomer of such an Fc fusion protein includes an extracellular region of a TIM linked to a constant heavy chain or portion thereof (for example, hinge, CH2, CH3 domains) of an immunoglobulin. The constant heavy chain in certain embodiments can include part or all of the CHl domain that is N-terminal to the hinge region of an immunoglobulin. In other embodiments, the constant heavy chain can include the hinge but not the CHl domain. In yet another embodiment, the constant heavy chain will exclude the hinge and the CHl domain, for example, it will include only the CH2 and CH3 domains of IgG.

[0041] In one embodiment, the TIM-I ligand targeting molecule can be a TIM-4 antibody. In another embodiment, the TIM-I ligand targeting molecule is a TIM-I-Fc fusion polypeptide. One skilled in the art can readily make a variety of TIM fusion polypeptides to an Fc or other desired polypeptide, including TIM polypeptide fragments containing desired domains.

[0042] Targeting occurs when an agent or TIM targeting molecule directly or indirectly binds to, or otherwise interacts with, a TIM or TIM ligand or a component of a TIM or TIM ligand signaling pathway in a way that affects the activity of the TIM or TIM ligand. Activity can be assessed by those of ordinary skill in the art and with routine laboratory methods (see, for example, Reith, Protein Kinase Protocols Humana Press, Totowa NJ (2001); Hardie, Protein Phosphorylation: A Practical Approach second ed., Oxford University Press, Oxford, United Kingdom (1999); Kendall and Hill, Signal Transduction Protocols: Methods in Molecular Biology Vol. 41, Humana Press, Totowa NJ (1995)). For example, one can assess the strength of signal transduction or another downstream biological event that occurs, or would normally occur, following receptor binding. The activity generated by an agent that targets a TIM or TIM ligand can be, but is not necessarily, different from the activity generated when a naturally occurring TIM or TIM ligand binds a naturally occurring TIM or TIM ligand.

[0043] As described above, agents of the invention can contain two functional moieties: a targeting moiety that targets the agent to a TIM-I ligand or TIM-I ligand- bearing cell and, for example, a dimerizing and/or target-cell depleting moiety that, for example, lyses or otherwise leads to the elimination of the TIM-I or TIM-I ligand- bearing cell, as discussed herein. Thus, the agent can be a chimeric polypeptide that includes a TIM-I polypeptide fragment that binds to a TIM-I ligand and a heterologous polypeptide such as the Fc region of the IgG and IgM subclasses of antibodies. The Fc region may include a mutation that inhibits complement fixation and Fc receptor binding, or it may be lytic or target-cell depleting, that is, able to destroy cells by binding complement or by another mechanism, such as antibody- dependent complement lysis. Accordingly, the Fc can be lytic and can activate complement and Fc receptor-mediated activities, leading to target cell lysis, allowing depletion of desired cells that express a TIM or TIM ligand.

[0044] The Fc region can be isolated from a naturally occurring source, recombinantly produced, or chemically synthesized using well known methods of peptide synthesis. For example, an Fc region that is homologous to the IgG C terminal domain can be produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The polypeptides of the invention can include the entire Fc region, or a smaller portion that retains the ability to lyse cells. In addition, full-length or fragmented Fc regions can be variants of the wild type molecule, that is, they can contain mutations that may or may not affect the function of the polypeptide. The Fc region can be derived from an IgG, such as human IgGl, IgG2, IgG3, IgG4, or analogous mammalian IgGs or from an IgM, such as human IgM or analogous mammalian IgMs. In a particular embodiment, the Fc region includes the hinge, CH2 and CH3 domains of human IgGl or IgG4 or murine IgG2a.

[0045] The Fc region that can be part of the TIM-I ligand targeting molecules or agents of the invention can be "target-cell depleting," also referred to herein as lytic, or "non target-cell depleting," also referred to herein as non-lytic. A non target-cell depleting Fc region typically lacks a high affinity Fc receptor binding site and a C¹Iq binding site. The high affinity Fc receptor binding site of murine IgG Fc includes the Leu residue at position 235 of IgG Fc. Thus, the murine Fc receptor binding site can be destroyed by mutating or deleting Leu 235. For example, substitution of GIu for Leu 235 inhibits the ability of the Fc region to bind the high affinity Fc receptor. The murine CIq binding site can be functionally destroyed by mutating or deleting the GIu 318, Lys 320, and Lys 322 residues of IgG. For example, substitution of Ala residues for GIu 318, Lys 320, and Lys 322 renders IgGl Fc unable to direct antibody-dependent complement lysis. In contrast, a target-cell depleting IgG Fc region has a high affinity Fc receptor binding site and a CIq binding site and can reduce the amount of target cell, for example, by Fc lytic activity or other mechanisms, as disclosed herein. The high affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, and the CIq binding site includes the GIu 318, Lys 320, and Lys 322 residues of IgGl . Target-cell depleting IgG Fc has wild type residues or conservative amino acid substitutions at these sites. Target- cell depleting IgG Fc can target cells for antibody dependent cellular cytotoxicity or complement directed cytolysis (CDC). Appropriate mutations for human IgG are also known (see, for example, Morrison et al., The Immunologist 2:119-124 (1994); and Brekke et al., The Immunologist 2:125, 1994). One skilled in the art can readily determine analogous residues for the Fc region of other species to generate target-cell depleting or non target-cell depleting fusions with a TIM targeting molecule or agent.

[0046] The compositions and methods of the invention can additionally be used to treat autoimmune diseases, for example, multiple sclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, alopecia areata, or other autoimmune disorders. Autoimmune diseases are a class of diseases in which a subject's own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self-peptides and cause destruction of tissue. Thus, an immune response is mounted against a subject's own antigens, referred to as self-antigens. Autoimmune diseases include the examples described above and also Crohn's disease and other inflammatory bowel diseases such as ulcerative colitis, systemic lupus erythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (for example, pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, autoimmune-associated infertility, glomerulonephritis (for example, crescentic glomerulonephritis, proliferative glomerulonephritis), bullous pemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance, autoimmune diabetes mellitus (type I diabetes mellitus; insulin-dependent diabetes mellitus), autoimmune hepatitis, autoimmune hemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune uveoretinitis, and Guillain-Barre syndrome. Recently, autoimmune disease has been recognized also to encompass atherosclerosis and Alzheimer's disease. A self- antigen refers to an antigen of a normal host tissue. Normal host tissue does not include cancer cells. Thus, an immune response mounted against a self-antigen, in the context of an autoimmune disease, is an undesirable immune response and contributes to destruction and damage of normal tissue, whereas an immune response mounted against a cancer antigen is a desirable immune response and contributes to destruction of the tumor or cancer. [0047] As disclosed herein, alopecia areata (AA) is a chronic autoimmune disease characterized by spontaneous reversible inflammation of the hair follicles. AA is thought to occur in about 1.7% of the population, or about 4.7 million people in the United States (Tang et al., J. Invest. Dermatol. 8:212-216 (2003). In humans, the disease can affect men, women, and children and cause hair loss (alopecia) in distinct patches or can occur diffusely on the scalp. Approximately 7% of affected individuals can have total loss of body or scalp hair. Current treatment of AA consists of topical and oral corticosteroids, which target the inflammation that occurs. The inflammation consists primarily of lymphocytes. In animal models of AA, which exhibit characteristics of human disease, the cytokine profile is primarily that of a ThI phenotype (Carroll et al., J. Invest. Dermatol. 119:392-402 (2002); Gilhar et al., Clin. Immunol. 106:181-187 (2003)). One such model uses aged C3H/HeJ mice that spontaneously develop disease in 20% of animals. Transfer of full thickness skin grafts from affected mice onto unaffected recipients results in 100% of animals with the AA phenotype (McElwee et al., J. Invest. Dermatol. 111 :797-803 (1998)). The pathogenesis of AA in the recipient mice is poorly understood, but thought to involve both CD4+ and CD8 + lymphocytes, as well CD4- CD25+ T cells (Gilhar et al., Arch. Dermatol. 138:916-922 (2002).

[0048] Recent studies have described a new class of immune regulating molecules called the TIMs (Moss et al., Expert Opin. Biol. Ther. 4:1887-1896 (2004)). TIM-I, for example, has been described to be a key regulator of Thl/Th2 immunity. The TIM genes were originally described as being closely linked to genes that control airway hyperreactivity, characteristic of asthma (Mclntire et al., Nat. Immunol. 2: 1109-16 (2001)). In humans, certain polymorphisms of the TIM-I gene appear to be associated with protection from asthma and atopy (Gao et al., J. Allergy Clin. Immunol. 115:982-988 (2005)). In animal models, antibodies and fusion proteins to TIM-I appear to have either ThI or Th2 stimulating effects. For example, in a murine model of asthma, antibodies to TIM-I appear to significantly decrease airway inflammation and also decrease Th2 cytokines (Encinas et al., J. Allergy Clin. Immunol. 116:1343-1349 (2005)). In other studies, antibodies to TIM-I were found to stimulate both ThI and Th2 cytokine production when administered in the presence of antigen (Umetsu et al., Nat. Immunol. 6:447-454 (2005). Furthermore, TIM-I fusion protein containing both the extracellular Ig and mucin domains of TIM-I, when administered to SJL/J mice immunized with a peptide of proteolipid protein, increases Th2 cytokines (Meyers et al., Nat. Immunol. 6:455-464 (2005)). This same TIM-I fusion protein containing the extracellular Ig and mucin domains of TIM-I fused to the Fc domains of an IgG molecule does not have any effects on disease development or progression in animal models of autoimmunity or transplant rejection.

[0049] The results disclosed herein are the first to indicate that TIM-I/ Fc, containing the extracellular Ig domain of TIM-I fused to the Fc domains of an IgG molecule, has an effect on sparing hair loss in an alopecia areata mouse model (see Example V). Thus, TIM-l/Fc, containing the extracellular Ig domain of TIM-I fused to the Fc domains of an IgG molecule, offers a method to treat alopecia areata and other autoimmune diseases.

[0050] The pathogenesis of AA is poorly understood, but thought to involve lymphocytic inflammation of the hair follicles, with concomitant hair loss. In animal models, some studies suggest that ThI type inflammation may play an important role in this disease. For example, the disease itself can be induced by interferon -gamma, a dominant ThI cytokine, or reduced by decreases in IL-2 in small animals (Freyschmidt- Paul et al., J. Invest. Dermatol. 125:945-951 (2005); Gilhar et al., J. Invest. Dermatol. 124:288-289 (2005)). The disease in animals and humans can also be precipitated by transfer of CD4 + and CD8+ T lymphocytes (McElwee et al., J. Invest. Dermatol. 119:1426-1433 (2002)). As disclosed herein, the model of full thickness skin grafts from alopecic C3H/HeJ mice to recipient mice was utilized to test the effects of TIM-l/Fc on the development of hair loss. This model resulted in alopecia in all of the recipient mice. However, administration of TIM-l/Fc resulted in significantly less alopecia compared to mice who had received an Ig control. In addition, mice that received TIM-l/Fc had significantly stronger in vitro lymphocyte proliferation and production of Th2, but not ThI type cytokines in response to Con A stimulation in vitro.

[0051] Clinically, alopecia areata can be treated with non-specific immunomodulators such as corticosteroids. In animals, however, previous reports suggest that inhibiting ThI and Delayed Type Hypersensitivity (DTH) inflammation can ameliorate AA. For example, in a C3H/HeJ model, treatment of animals with an antibody to CD44 splice variant 10, which previously had been shown to inhibit DTH, also inhibited the onset and extent of AA (Freyschmidt-Paul et al., J. Invest. Dermatol. 115:653-657 (2000)). [0052] The TIM gene family of molecules has recently been described as modulators of Thl/Th2 immunity and offers the potential to bi-directionally modulate the balance of the immune system. One ligand for TIM-I has been described to be TIM-4 (Meyers et al., Nat. Immunol. 6:455-464 (2005)). Initial observations suggested that antibody to TIM-I may down regulate Th2 mediated diseases such as asthma (Encinas et al., J. Allergy Clin. Immunol. 116: 1343-1349 (2005)). However, administration of TIM- 1/Fc resulted in enhanced lymphocyte proliferation with the production of Th2 cytokines. Therefore, TIM-I/ Fc has therapeutic effects in alopecia areata (see Example V).

[0053] Thus, the invention also provides a method for ameliorating a sign or symptom associated with alopecia areata by administering a TIM-I ligand targeting molecule to a subject, wherein the TIM-I ligand targeting molecule is dimeric. As disclosed herein, the effects of TIM-l/Fc were examined in a murine model of alopecia areata. Full thickness skin grafts were performed from donor affected C3H/HeJ mice onto unaffected animals, and all recipient animals developed alopecia. Animals were treated with 100 μg intraperitoneally of TIM-l/Fc, or an IgG control at the onset of alopecia. By days 49-51 post skin grafting, animals that received TIM-l/Fc had a significantly smaller area of alopecia compared to the IgG treated control group (P<0.05). Furthermore, the TIM-l/Fc treated group had stronger in vitro concanavalin A (Con A) stimulated lymphocyte proliferation compared to the IgG treated control group (P<0.05). In vitro production of multiple Th2 cytokines was also augmented in the TIM-l/Fc treated group compared to the IgG control group. The results disclosed herein are the first to indicate that TIM-l/Fc has an effect on sparing hair loss in a murine model of alopecia areata. Furthermore, TIM-l/Fc was observed to increase lymphocyte proliferation and Th2 cytokines. Thus, TIM-l/Fc offers can be used to treat alopecia areata and other autoimmune diseases.

[0054] It is understood that the compositions and methods of the invention can be combined with other therapies for treating a particular condition. For example, the use of a composition of the invention for treating autoimmune diseases can be optionally combined with therapies used to treat a particular autoimmune disease.

[0055] In an additional embodiment, the invention provides the use of a composition comprising a TIM-I ligand targeting molecule or agent conjugated to a therapeutic moiety such as an immunotoxin for the manufacture of a medicament for treating an autoimmune disorder in a subject. In yet a further embodiment, the invention provides the use of a TIM targeting molecule or agent conjugated to a therapeutic moiety where the autoimmune disorder is a disorder selected from rheumatoid arthritis, multiple sclerosis, autoimmune diabetes mellitus, systemic lupus erythematosus, autoimmune lymphoproliferative syndrome (ALPS), and the like.

[0056] As disclosed herein, a dimeric TIM-I ligand targeting molecule has been shown to be effective at ameliorating signs and/or symptoms associated with two representative autoimmune diseases, a mouse model of multiple sclerosis (Example IV) and a mouse model of alopecia areata (Example V). Thus, dimeric TIM-I ligand targeting molecules have been demonstrated to be effective at decreasing an immune response in autoimmune diseases. A dimeric TIM-I ligand targeting molecule can similarly be used to decrease an immune response in transplant rejection, in which suppression of an immune response against a transplanted tissue is desired.

[0057] Thus, the invention additionally provides a method for ameliorating a sign or symptom associated with transplant rejection by administering a TIM-I ligand targeting molecule to a subject, wherein the TIM-I ligand targeting molecule is dimeric. The TIM-I ligand targeting molecule can be, for example, a TIM-4 antibody. The Fc portion of the TIM-4 antibody can be target-cell depleting or non target-cell depleting. The TIM-I ligand targeting molecule can also be a TIM-I-Fc fusion polypeptide. The Fc portion of the TIM-I-Fc fusion polypeptide can be target-cell depleting or non target-cell depleting. For example, a TIM-I ligand targeting molecule can be a TIM-I-Fc fusion polypeptide containing a TIM-I Ig domain, in particular in the absence of a TIM-I mucin domain.

[0058] The methods of the invention can be used to treat various types of transplants, for example, a transplanted cell, tissue or organ. Transplants also include bone marrow grafts, graft versus host disease, as well as cell transplants such as islet cells or grafting of cells producing a therapeutic polypeptide, as disclosed herein, for example, insulin-producing pig pancreatic cells or cells engineered to express a therapeutic polypeptide. Transplants also include skin allografts. These and other types of transplanted cells, tissues or organs can be treated to decrease rejection or other signs or symptoms associated with a transplant using a dimeric TIM-I ligand targeting molecule. [0059] The invention further provides a fusion polypeptide comprising a TIM-I polypeptide fragment containing the TIM-I IgG fragment fused to a Fc. Exemplary fusion polypeptides are described in Examples I and II. For example, a TIM-I ligand targeting molecule can be a TIM-I-Fc fusion polypeptide containing a TIM-I Ig domain, in particular in the absence of a TIM-I mucin domain. One skilled in the art can readily generate similar fusion proteins, as disclosed herein. The invention additionally provides a pharmaceutical composition comprising a fusion polypeptide comprising a TIM-I polypeptide fragment containing the TIM-I IgG fragment fused to a Fc in a pharmaceutical carrier.

[0060] The invention also provides a TIM-4 antibody, including monoclonal and polyclonal antibodies. In a particular embodiment, the TIM-4 antibody is specific for the extracellular domain of TIM-4, in particular the immunoglobulin domain of TIM-4 and/or the mucin domain (see Example III). Additionally provided is a pharmaceutical composition comprising a TIM-4 antibody in a pharmaceutical carrier.

[0061] Compositions of the invention can be administered locally or systemically by any method known in the art, including, but not limited to, intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. Additional routes include intracranial (for example, intracisternal or intraventricular), intraorbital, opthalmic, intracapsular, intraspinal, and topical administration. The compositions of the invention can be administered in a suitable, nontoxic pharmaceutical carrier, or can be formulated in microcapsules or as a sustained release implant. The immunogenic compositions of the invention can be administered multiple times, if desired, in order to sustain the desired immune response. The appropriate route, formulation and immunization schedule can be determined by those skilled in the art.

[0062] A TIM targeting molecule can be administered in one or more different forms. If the TIM targeting molecule is a peptide or polypeptide, such as an anti-TIM antibody or a TIM fusion protein, modes of administration include, but are not limited to, administration of the purified peptide or polypeptide, administration of cells expressing the peptide or polypeptide, or administration of nucleic acids encoding the peptide or polypeptide. [0063] The methods of the present invention and the therapeutic compositions used to carry them out contain "substantially pure" agents. For example, in the event the TIM targeting molecule or agent is a polypeptide, the polypeptide can be at least about 60% pure relative to other polypeptides or undesirable components in the original source of the polypeptide. For example, if a polypeptide is purified from a natural source, from recombinant expression, or chemical synthesis, the purity is relative to other components in the original natural source, recombinant source, or synthetic reaction. One skilled in the art can readily determine appropriate well known purification methods for a polypeptide agent or other agents of the invention. In particular, the agent can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99% purity, or higher purity, as desired. One skilled in the art can readily determine a suitable purity for a particular desired application. Purity can be measured by any appropriate standard method, for example, column chromatography, polyacrylamide gel electrophoresis, HPLC analysis, and can be based on desired quantification criteria such as ultraviolet absorbance, staining, or similar methods of measuring quantities depending on the chemical nature of the agent. It is understood that when an agent of the invention is combined with other components that the TIM targeting molecule or agent can be administered at a particular purity, for example 95% purity, but is not required to be 95% of the total components. One skilled in the art can readily determine a suitable purity and a suitable amount of the TIM targeting molecule or agent relative to other desirable components in a composition of the invention.

[0064] Although agents useful in the methods of the present invention can be obtained from naturally occurring sources, they can also be synthesized or otherwise manufactured, for example, by expression of a recombinant nucleic acid molecule encoding a TIM-I ligand targeting molecule or agent. Methods for recombinantly expressing polypeptides are well known to those skilled in the art (Ausubel et al., Current Protocols in Molecular Biology (Supplement 56), John Wiley & Sons, New York (2001); Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001)). Methods of peptide synthesis are also well known to those skilled in the art (Merrifield, J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, Principles of Peptide Synthesis Springer-Verlag (1984)). Polypeptides that are purified from a natural source, for example, from eukaryotic organisms, can be purified to be substantially free from their naturally associated components. Similarly, polypeptides that are expressed recombinantly in eukaryotic or prokaryotic cells, for example, E. coli or other prokaryotes, or that are chemically synthesized can be purified to a desired level of purity. In the event the polypeptide is a chimera, it can be encoded by a hybrid nucleic acid molecule containing one sequence that encodes all or part of the agent, for example, a sequence encoding a TIM polypeptide and sequence encoding an Fc region of IgG.

[0065] Agents of the invention, in particular, polypeptides expressed recombinantly, can be fused to an affinity tag to facilitate purification of the polypeptide. In one embodiment, the affinity tag can be a relatively small molecule that does not interfere with the function of the polypeptide, for example, binding of a TIM targeting molecule or agent. Alternatively, the affinity tag can be fused to a polypeptide with a protease cleavage site that allows the affinity tag to be removed from the recombinantly expressed polypeptide. The inclusion of a protease cleavage site is particularly useful if the affinity tag is relatively large and could potentially interfere with a function of the polypeptide. Exemplary affinity tags include a poly-histidine tag, generally containing about 5 to about 10 histidines, or hemagglutinin tag, which can be used to facilitate purification of recombinantly expressed polypeptides from prokaryotic or eukaryotic cells. Other exemplary affinity tags include maltose binding protein or lectins, both of which bind sugars, glutathione-S transferase, avidin, and the like. Other suitable affinity tags include an epitope for which a specific antibody is available. An epitope can be, for example, a short peptide of about 3-5 amino acids or more, a carbohydrate, a small organic molecule, and the like. Epitope tags have been used to affinity purify recombinant proteins and are commercially available. For example, antibodies to epitope tags, including myc, FLAG, hemagglutinin (HA), green fluorescent protein (GFP), polyHis, and the like, are commercially available (see, for example, Sigma, St. Louis MO; PerkinElmer Life Sciences, Boston MA).

[0066] In therapeutic applications, agents of the invention can be administered with a physiologically acceptable carrier, such as physiological saline. The therapeutic compositions of the invention can also contain a carrier or excipient, many of which are known to one of ordinary skill in the art. Excipients that can be used include buffers, for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer; amino acids; urea; alcohols; ascorbic acid; phospholipids; proteins, for example, serum albumin; ethylenediamine tetraacetic acid (EDTA); sodium chloride or other salts; liposomes; mannitol, sorbitol, glycerol, and the like. The agents of the invention can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for ingestion or injection; gels or powders can be made for ingestion, inhalation, or topical application. Methods for making such formulations are well known and can be found in, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton PA (1990).

[0067] The invention additionally provides the use of compositions of the invention in preparation of a medicament to treat an autoimmune disease or transplant rejection by alleviating a sign or symptom associated with the autoimmune disease or transplant rejection. In particular, the invention provides the use of a TIM-I ligand targeting molecule, for example, a TIM-4 antibody or TIM-I-Fc fusion polypeptide in preparation of a medicament to treat an autoimmune disease or transplant rejection by alleviating a sign or symptom associated with the autoimmune disease or transplant rejection.

[0068] As discussed above, polypeptide agents of the invention, including those that are fusion proteins, can be obtained by expression of one or more nucleic acid molecules in a suitable eukaryotic or prokaryotic expression system and subsequent purification of the polypeptide agents. In addition, a polypeptide agent of the invention can also be administered to a patient by way of a suitable therapeutic expression vector encoding one or more nucleic acid molecules, either in vivo or ex vivo. Furthermore, a nucleic acid can be introduced into a cell of a graft prior to transplantation of the graft. Thus, nucleic acid molecules encoding the agents described above are within the scope of the invention.

[0069] Just as polypeptides of the invention can be described in terms of their identity with wild type polypeptides, the nucleic acid molecules encoding them will have a certain identity with those that encode the corresponding wild type polypeptides. For example, the nucleic acid molecule encoding TIM-I, TIM-2, TIM-3 or TIM-4 can be at least about 50%, at least about 65%, at least about 75%, at least 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to the nucleic acid encoding natural or wild-type TIM-I, TIM-2, TIM-3 or TIM-4. Similarly, the TIM polypeptides can have at least about 50%, at least about 65%, at least about 75%, at least 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identical to the natural or wild-type TIM-I, TIM-2, TIM-3 or TIM-4 polypeptides. It is understood that a polypeptide or encoding nucleic acid that has less than 100% identity with a corresponding wild type molecule still retains a desired function of the TIM polypeptide.

[0070] The nucleic acid molecules that encode agents of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA, for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis, or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double stranded or single stranded, either a sense or an antisense strand. It is understood by those skilled in the art that a suitable form of nucleic acid can be selected based on the desired use, for example, expression using viral vectors that are single or double stranded and are sense or antisense.

[0071] In the case of a naturally occurring nucleic acid molecule of the invention, the nucleic acid molecule can be "isolated" from the naturally occurring genome of an organism because they are separated from either the 5' or the 3' coding sequence with which they are immediately contiguous in the genome. Thus, a nucleic acid molecule includes a sequence that encodes a polypeptide and can include non-coding sequences that lie upstream or downstream from a coding sequence. Those of ordinary skill in the art are familiar with routine procedures for isolating nucleic acid molecules (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, New York (1989); Ausubel et al., Current Protocols in Molecular Biology (Supplement 56), John Wiley & Sons, New York (2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001)). The nucleic acid can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR) to amplify a desired region of genomic DNA or cDNA using well known methods (see, for example, Dieffenbach and Dveksler, PCR Primer: A Laboratory Manual Cold Spring Harbor Press (1995)). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced by in vitro transcription.

[0072] The isolated nucleic acid molecules of the invention can include fragments not found in the natural state. Thus, the invention encompasses recombinant molecules, such as those in which a nucleic acid sequence, for example, a sequence encoding TIM-I, TIM-2 TIM-3 or TIM-4, is incorporated into a vector, for example, a plasmid or viral vector, or into the genome of a heterologous cell or the genome of a homologous cell, at a position other than the natural chromosomal location.

[0073] As described above, agents of the invention can be fusion proteins. In addition to, or in place of, the heterologous polypeptides described above, a nucleic acid molecule encoding an agent of the invention can contain sequences encoding a "marker" or "reporter." Examples of marker or reporter genes include β lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo^r, G418^r), dihydro folate reductase (DHFR), hygromycin B- phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associated with the practice of the invention, one of ordinary skill in the art will be aware of additional useful reagents, for example, of additional sequences that can serve the function of a marker or reporter.

[0074] Nucleic acid molecules encoding a TIM-I, TIM-2, TIM-3 or TIM-4 molecule can be obtained from any biological cell, such as the cell of a mammal, or produced by routine cloning methods. Thus, the nucleic acids of the invention can be those of a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In a particular embodiment, the nucleic acid molecules can encode a human TIM.

[0075] A nucleic acid molecule of the invention described herein can be contained within a vector that is capable of directing its expression in, for example, a cell that has been transduced with the vector. Accordingly, in addition to polypeptide agents, expression vectors containing a nucleic acid molecule encoding those agents and cells transfected with those vectors are provided.

[0076] Vectors suitable for use in the present invention include T7 based vectors for use in bacteria (see, for example, Rosenberg et al., Gene 56: 125-135 (1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521-3527 (1988), yeast expression systems, such as Pichia pastoris , for example the PICZ family of expression vectors (Invitrogen, Carlsbad, CA) and baculovirus derived vectors, for example the expression vector pBacPAK9 (Clontech, Palo Alto, CA) for use in insect cells. The nucleic acid inserts, which encode the polypeptide of interest in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which the nucleic acid is to be expressed. For example, a T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue specific and cell type specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body. One of ordinary skill in the art can readily determine a suitable promoter and/or other regulatory elements that can be used to direct expression of nucleic acids in a desired cell or organism.

[0077] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neo^r) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Other feasible selectable marker genes allowing for phenotypic selection of cells include various fluorescent proteins, for example, green fluorescent protein (GFP) and variants thereof. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for a particular use. An exemplary vector is shown in Figure 1.

[0078] Viral vectors that can be used in the invention include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukarvotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, New York).

[0079] Prokaryotic or eukaryotic cells that contain a nucleic acid molecule that encodes an agent of the invention and that express the protein encoded in the nucleic acid molecule are also provided. A cell of the invention is a transfected cell, that is, a cell into which one or more nucleic acid molecules encoding a molecule of interest such as a TIM- 1 ligand targeting molecule or for example nucleic acids encoding for the heavy and light chains of an anti-TIM-4 antibody, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the invention. A variety of expression systems can be utilized. For example, a TIM-I, TIM- 2, TIM-3 or TIM-4 or anti-TIM-1, anti-TIM-2, anti-TIM-3 or anti-TIM-4 polypeptides can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell, for example, Sf21 cells, or mammalian cells, for example, COS cells, CHO cells, 293 cells, PER.C6 cells, NIH 3T3 cells, HeLa cells, and the like. These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). One skilled in the art can readily select appropriate components for a particular expression system, including expression vector, promoters, selectable markers, and the like, as discussed above, suitable for a desired cell or organism. The selection and use of various expression systems can be found, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY (1993); and Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987). Also provided are eukaryotic cells that contain a nucleic acid molecule encoding an agent of the invention and express the protein encoded by such a nucleic acid molecule.

[0080] Furthermore, eukaryotic cells of the invention can be cells that are part of a cellular transplant, a tissue or organ transplant. Such transplants can comprise either primary cells taken from a donor organism or cells that were cultured, modified and/or selected in vitro before transplantation to a recipient organism, for example, eurkaryotic cells lines, including stem cells or progenitor cells. If, after transplantation into a recipient organism, cellular proliferation occurs, the progeny of such a cell are also considered within the scope of the invention. A cell, being part of a cellular, tissue or organ transplant, can be transfected with a nucleic acid encoding a TIM or anti-TIM polypeptide and subsequently be transplanted into the recipient organism, where expression of the polypeptide occurs. Furthermore, such a cell can contain one or more additional nucleic acid constructs allowing for application of selection procedures, for example, of specific cell lineages or cell types prior to transplantation into a recipient organism. Such transplanted cells can be used in therapeutic applications. For example, if the TIM targeting molecule or agent is a polypeptide, cells expressing the TIM targeting molecule can be transplanted to provide a source of the TIM targeting molecule using well known methods of gene delivery and suitable vectors (see, for example, Kaplitt and Loewy, Viral Vectors: Gene Therapy and Neuroscience Applications Academic Press, San Diego (1995)).

[0081] In the case of cell transplants, the cells can be administered either by an implantation procedure or with a catheter-mediated injection procedure through the blood vessel wall. In some cases, the cells may be administered by release into the vasculature, from which the cells subsequently are distributed by the blood stream and/or migrate into the surrounding tissue.

[0082] In another embodiment, a TIM targeting molecule that functions as an immunosuppressive agent can be introduced by gene delivery methods to cells of the organ. In such a case, the donor organ itself provides an immunosuppressive agent to facilitate organ transplant and inhibit transplant rejection.

[0083] As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. An antibody specific for an antigen, or an antigen binding fragment of such an antibody, is characterized by having specific binding activity for an antigen or an epitope thereof of at least about IxIO⁵M^"1. Thus, Fab, F(ab')₂, Fd and Fv fragments of an antibody specific for an antigen, which retain specific binding activity for an antigen, are included within the definition of an antibody. Specific binding activity to an antigen such as a TIM can be readily determined by one skilled in the art, for example, by comparing the binding activity of an antibody to its respective antigen versus a non-antigen control molecule. One skilled in the art will readily understand the meaning of an antibody having specific binding activity for a particular antigen, for example, a TIM. The antibody can be a polyclonal or a monoclonal antibody. Methods of preparing polyclonal or monoclonal antibodies are well known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)). When using polyclonal antibodies, the polyclonal sera can be affinity purified using the antigen to generate mono-specific antibodies having reduced background binding and a higher proportion of antigen-specific antibodies.

[0084] In addition, the term "antibody" as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen- binding fragments thereof. Humanized antibodies are meant to include recombinant antibodies generated by combining human immunoglobulin sequences, for example, human framework sequences, with non-human immunoglobulin sequences derived from complementarity determining regions (CDRs) providing antigenic specificity. Non- human immunoglobulin sequences can be obtained from various non-human organisms suitable for antibody production, including but not limited to rat, mouse, rabbit goat, and the like. Humanized antibodies are also meant to include fully human antibodies. Methods for obtaining fully human antibodies, such as using for example phage display library systems or human MHC locus transgenic mice, are well known in the art (see, for example, U.S. Patent Nos. 5,585,089; 5,530,101 ; 5,693,762; 6,180,370; 6,300,064; 6,696,248; 6,706,484; 6,828,422; 5,565,332; 5,837,243; 6,500,931 ; 6,075,181 ; 6,150,584; 6,657,103; 6,162,963). Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al. (Science 246:1275-1281 (1989)). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989) ; Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995)).

[0085] Antibodies specific for an antigen can be raised using an immunogen such as an isolated TIM polypeptide, or a fragment thereof, which can be prepared from natural sources or produced recombinantly, or an antigenic portion of the antigen that can function as an epitope. Such epitopes are functional antigenic fragments if the epitopes can be used to generate an antibody specific for the antigen. A non-immunogenic or weakly immunogenic antigen or portion thereof can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art (see, for example, Harlow and Lane, supra, 1988). An immunogenic peptide fragment of an antigen can also be generated by expressing the peptide portion as a fusion protein, for example, to glutathione S transferase (GST), polyHis, or the like. Methods for expressing peptide fusions are well known to those skilled in the art (Ausubel et al, Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)).

[0086] A TIM targeting molecule can be expressed recombinantly, as disclosed herein, as a polypeptide, a functional fragment of a polypeptide having a desired activity, or as a fusion polypeptide. Methods of making and expressing recombinant forms of a TIM targeting molecule are well known to those skilled in the art, as taught, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, New York (1989); Ausubel et al., Current Protocols in Molecular Biology (Supplement 56), John Wiley & Sons, New York (2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001). Such methods are exemplified in the Examples, and Figure 1 shows an exemplary expression vector for a TIM-I ligand targeting molecule construct. One skilled in the art can readily determine a desired fragment, for example, a functional fragment of a TIM having a desired function, for example, the extracellular domain or a fragment thereof such as the Ig domain and/or mucin domain, for use as a TIM targeting molecule.

[0087] As discussed above, a TIM targeting molecule or agent can be a small molecule, a peptide, a polypeptide, a polynucleotide, including antisense and siRNAs, a carbohydrate including a polysaccharide, a lipid, a drug, as well as mimetics, and the like. Methods for generating such molecules are well known to those skilled in the art (Huse, U.S. Patent No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, MoI. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); Gordon et al., J. Med. Chem. 37: 1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); Gordon et al., Ace. Chem. Res. 29: 144-154 (1996); Wilson and Czarnik, eds., Combinatorial Chemistry: Synthesis and Application, John Wiley & Sons, New York (1997)). Methods for selecting and preparing antisense nucleic acid molecules are well known in the art and include in silico approaches (Patzel et al., Nucl. Acids Res. 27:4328-4334 (1999); Cheng et al., Proc. Natl. Acad. Sci. USA 93:8502-8507 (1996); Lebedeva and Stein, Ann. Rev. Pharmacol. Toxicol. 41 :403-419 (2001); Juliano and Yoo, Curr. Opin. MoI. Ther. 2:297-303 (2000); and Cho-Chung, Pharmacol. Ther. 82:437-449 (1999)). Methods for producing si RNAs and using RNA interference have been described previously (Fire et al., Nature 391 :806- 811 (1998); Hammond et al. Nature Rev. Gen. 2: 110-119 (2001); Sharp, Genes Dev. 15: 485-490 (2001); and Hutvagner and Zamore, Curr. Opin. Genetics & Development 12:225-232( 2002); Hutvagner and Zamore, Curr. Opin. Genetics & Development 12:225- 232 (2002); Bernstein et al., Nature 409:363-366 (2001); (Nykanen et al., CeU 107:309- 321 (2001)).

[0088] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Generation of TIM-l/Fc expression vectors

[0089] A shuttle plasmid vector (pMGDNA) was used for the cloning and expression of TIM-l/Fc fusion proteins. The pMGDNA plasmid vector is a mammalian expression vector designed for the secretion, purification, and detection of recombinant proteins (see Figure 1). The basic vector has a multiple cloning site that is used to insert the gene of interest to be expressed and some or all of the following features: cytomegalovirus (CMV) enhancer/promoter and 5 '-untranslated intron A for high-level constitutive expression; Kozac consensus sequence (GCCACC) prior to the initiating ATG of the human CD5 signal sequence; human CD5 secretion signal sequence for efficient secretion of fusion proteins; multiple cloning sites; human beta globin polyadenylation signal and transcription termination sequence to enhance mRNA stability; col El Ori region for plasmid replication; SV40 enhancer and early promoter for expression of neomycin resistance gene; SV40 minimum origin of replication; coding sequence for a chimeric kanamycin/neomycin resistance gene, which allows selection of transfected E. coli with kanamycin and transfected mammalian cells with G418; and HSV tk polyadenylation signal.

[0090] Fragments of the mouse and human TIM-I genes and Ig Fc gene fragments were cloned in frame with the N-terminal hCD5 leader sequence into pMGDNA, to yield the resulting TIM-l/Fc fusion protein gene. The accuracy of all plasmid constructs was confirmed by DNA sequencing. The details of the cloning are described below in more detail. [0091] Cloning of human CD5 leader into pMGDNA vector. A human CD5 leader sequence gene fragment was synthesized using two oligonucleotides (Figure 7). The forward oligonucleotide (5' to 3')

TGGCACCGGTGCCACCATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTA CCTGCTGGGG (SEQ ID NO:45) and the reverse oligonucleotide (5' to 3') TAGGAGATCTCCTAGGCAGGAAGCGACCAGCATCCCCAGCAGGTACAAGGTGG CCAGCGG (SEQ ID NO:46) were used for the synthesis of the human CD5 leader. The two oligonucleotides were annealed to each other, filled-in by PCR reaction and cloned into the pMGDNA vector. The resulting pMGDNA.hCD5 vector was identified and confirmed by DNA sequencing.

[0092] Cloning of lytic and non-lvtic mouse IgG2a Fc into pMGDNA.hCD5. The mouse IgG2a Fc fragment can be amplified from mouse hybridomas expressing IgG2a immunoglobulins. A non-lytic mouse IgG2a Fc can subsequently be generated by site directed PCR mutagenesis or other methods known to the art. Exemplary sequences are shown in Figure 12.

[0093] Both lytic and nonlytic mouse IgG2a Fc genes share the same 5' and 3' sequences, and were amplified using the following PCR primer set: (a) mFc2a- 5' PCR primer (5' to 3'), 38 mer: 5'-

CTCTGGGATCCCAGAGGGCCCACAATCAAGCCCTGTCC -3' (BamHI site underlined; SEQ ID NO:47); (b) mFc2a- 3'-PCR primer (3' to 5'), 38 mer (5'- GATGAACTAGTTCATCATTTACCCGGAGTCCGGGAGAA -3 ' (Spel site underlined; SEQ ID NO:48). The resulting PCR fragments were restriction digested using the BamHI and Spel restriction enzymes and cloned into pMGDNA.hCD5. The resulting vector contains the hinge, CH2 and CH3 domains of mouse IgG2a Fc.

[0094] Cloning of human IgGl and IgG4 Fc into pMGDNA.hCD5. The human

IgGl (Figure 13) and IgG4 (Figure 14) Fc fragments can be amplified, for example, from B cell lines or lymphomas expressing IgGl and IgG4 immunoglobulins using specific PCR primers that contain suitable restriction enzyme cleavage sites and which will lead to the amplification of a PCR fragment encoding for the hinge, CH2 and CH3 domains of human IgGl and human IgG4. [0095] Cloning of TIM-I gene fragment into pMGDNA.hCD5Ig Fc vector to generate the final TIM-I /Fc expression vectors. The mouse and human TIM-I genes were amplified from suitable primary cells and cell lines expressing the respective TIM-I genes. The TIM-I gene is, for example, expressed in the kidney, and isolated kidney tissue can be used to amplify and clone the TIM-I genes. Other cells expressing TIM-I include various leukocytes, but also, for instance the mouse kidney adenocarcinoma cell line, RAG, and the human kidney adenocarcinoma cell line, 769P. Human TIM-I cDNA was amplified by PCR from 769P cells. Mouse TIM-I cDNA was amplified by PCR from RAG cells. Using TIM-I cDNA, TIM-I gene fragments of interest were further amplified by PCR and cloned into the pMGDNA.hCD5Ig Fc vectors to yield TIM-I /Fc expression vectors expressing both mouse and human TIM-I, fused to mouse and/or human IgFc fragments. The TIM-I part consisted of either the Ig or the Ig and mucin extracellular domains of the respective TIM-I . A schematic representation of a typical TIM-I /Fc expression vector is shown in Figure 1.

EXAMPLE II Purification of Dimeric TIM-l/Fc Fusion Proteins

[0096] The TIM-I /Fc expression vectors encoding for mouse and human TIM- l/Fc, with and without mucin domains, respectively, were transfected into Human Embryonic Kidney - 293 (HEK-293) cells. TIM-l/Fc fusion protein produced by the transfected cells and secreted into the culture supernatant was purified by Protein A (human TIM-l/Fc) or Protein G (mouse TIM-l/Fc) Sepharose™ affinity chromatography. In brief, supernatants from the transfected cells were concentrated, and the pH of the concentrated supernatants adjusted to pH 8.0 using diluted sodium hydroxide (NaOH) or 0.5 M sodium phosphate, pH 9.0. Subsequently, the supernatants were loaded onto a Protein A or G Sepharose column pre-equilibrated with 20 mM sodium phosphate, pH 8.0. The column was washed with 20 mM sodium phosphate, pH 8.0, and bound protein eluted with 100 mM Na-Citrate buffer, pH 3.5, into tubes containing 1.0 M Tris-HCl, pH 9.0. Peak protein fractions were combined and dialyzed against PBS. As necessary the protein was further purified using size exclusion chromatography.

[0097] Figure 15 shows an exemplary SDS-PAGE and Western blot of purified full length and mucinless TIM-l/Fc. Purified recombinant full length and mucinless TIM-l/Fc protein were separated on SDS-PAGE under reducing conditions and protein stained using Bio-Safe™ Coomassie stain (Biorad; Hercules CA). For Western blots, proteins separated by SDS-PAGE were transferred onto a polyvinylidene fluoride (PVDF) membrane. The transferred protein was then detected using an antibody against the IgG2a Fc portion of TIM-I /Fc fusion protein.

EXAMPLE III Generation of TIM-4 Monoclonal Antibodies

[0098] Expression vectors for mouse and human TIM-4/Fc were generated in a fashion analogous to TIM-l/Fc. As for TIM-l/Fc, constructs expressing the extracellular Ig domain only of TIM-4 and constructs expressing the extracellular Ig and mucin domains of TIM-4 were generated. For mouse TIM-4/Fc, the Fc portion (hinge, CH2, CH3 domains) of mouse IgG2a was cloned in-frame 3' of the TIM-4 portion. For human TIM-4/Fc, the Fc portion (hinge, CH2, CH3 domains) of human IgGl or human IgG4 was cloned in- frame 3' of the TIM-4 portion. The final expression vectors were used to either stably transfect Chinese hamster ovary (CHO) cells or to transiently transfect 293 cells. Tissue culture supernatants from transfected and TIM-4/Fc expressing cells were collected, concentrated, and TIM-4/Fc fusion protein purified as described in Example II.

[0099] To generate anti-mouse TIM-4 monoclonal antibodies, rats were subcutaneously immunized with mucinless or Ig and mucin domain containing mouse TIM-4 - mouse IgG2a Fc fusion proteins in Complete Freund's Adjuvant (CFA) followed by three subsequent immunizations with mouse TIM-4/Fc in incomplete Freund's adjuvant (IFA). During the course of the immunizations, serum anti-TIM-4 antibody titers were routinely checked by ELISA. Spleens from rats developing anti-TIM-4 immune responses were harvested, splenocytes prepared and fused with Yb2/0 cells. The hybridoma cells were expanded and screened by ELISA and flow cytometry using mouse TIM-4/Fc and control mouse IgG2a as antigen. The mouse TIM-4 specific clones with highest titers were selected and further subcloned by limiting dilution.

[00100] To generate anti-human TIM-4 monoclonal antibodies, mice were immunized with Ig and mucin domain containing TIM-4 - human IgGl Fc fusion proteins in Complete Freund's Adjuvant (CFA) followed by three subsequent immunizations with human TIM-4/Fc in incomplete Freund's adjuvant (IFA). During the course of the immunizations, serum anti-TIM-4 antibody titers were routinely checked by ELISA. Spleens from mice developing anti-TIM-4 immune responses were harvested, splenocytes prepared and fused with FO cells. The hybridoma cells were expanded and screened by ELISA and flow cytometry, using human TIM-4/Fc and control mouse IgG2a as antigen. The human TIM-4 specific clones with the highest titers were selected and further subcloned by limiting dilution.

EXAMPLE IV

Efficacy of Dimerized Mucinless TIM-l/Fc on Disease Course of Experimental Autoimmune Encephalomyelitis (EAE)

[00101] The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is thought to play a role in modulating autoimmune and atopic diseases. This example describes the effects of a dimerized form of mouse mucinless TIM-I /Fc fusion protein on the course of relapsing-remitting EAE in SJL/J mice.

[00102] The materials and methods and reagents used were as follows. The mouse mucinless TIM-IFc fusion protein (ML-mTIM-1/Fc) was prepared. The purified IgG2a (Rat anti mouse keyhole limpit hemocyanin (KLH), IgG2a isotype) control monoclonal antibody (Mab) was purchased from Pharmingen (San Diego CA). The proteolipid protein (PLP) PLP₁₃^_1S1 peptide was purchased from Multiple Peptide Systems (San Diego, CA). Pertussis Toxin (PT) and complete Freund's adjuvant (CFA) were purchased from List Biological (Campbell CA) and Sigma (St. Louis MO), respectively. The Thl/Th2 cytokine bead array kits for quantification of cytokines and the antibodies for flow cytometry were purchased from BD Biosciences (San Jose CA). The Delfia assay kit to measure proliferation was purchased from Perkin Elmer (Boston MA).

[00103] For the mouse TIM-I /Fc, the mucinless TIM-I /Fc consisted of the V- region devoid of the mucin domain that was fused to the non-lytic mouse IgG2a Fc tail.

[00104] For induction of EAE, the experiments were performed at Perry Scientific

(San Diego CA). The Perry Scientific Institutional Animal Care and Use Committee (IACUC) approved the protocol for the EAE studies. Six to eight weeks old female SJL/J mice were purchased from Jackson Laboratories (Bar Harbor ME). The EAE induction and ML-mTIM-1/Fc administration schedule is shown in Figure 2. Briefly, on day 0, mice were immunized with 100 μg Of PLP_{139-J 5I} peptide emulsified in CFA {Mycobacterium tuberculosis concentration at 200 μg) in the right and left flanks. Following the injection of the PLP₁₃₉₄₅₁ peptide in CFA, 100 ng of PT was injected intravenously via the tail vein. The animals were then monitored daily for disease symptoms. The disease was scored as follows: 0: no abnormality, healthy; 1 : limp tail or hind limb weakness; 2: limp tail and hind limb weakness; 3: limp tail and partial paralysis of hind or fore limbs; 4: limp tail and complete hind or fore limb paralysis or quadriplegia; and 5: death.

[00105] For administration of the proteins, fusion protein and control antibody were administered. Starting from day 8, mice were injected with 100 μg/dose of mTIM-lFc- ML or IgG2a intraperitoneally. A total of 3 injections at 48 hr intervals were administered. The animals were monitored for up to sixty days.

[00106] The effect of ML-mTIM-1/Fc on the acute phase of EAE was determined.

The data were derived from 3 independent experiments involving a total of 26 mice in each of the IgG2a control antibody treated and the ML-mTIM-1/Fc treated groups. The onset of EAE in SJL/J mice ranges from day 9 -13 and the mice gradually enter remission after approximately day 15-16. The acute phase usually ranges from day 11 to 15. Statistical analysis of the acute phase (days 10-15) data was performed by comparing the IgG2a treated mice with the ML-mTIM-1/Fc treated mice using the Mann- Whitney unpaired t-test. The parameters analyzed were the incidence, day of onset of the EAE symptoms and the maximum score achieved by the mice by day 15.

[00107] The incidence of EAE in mice treated with ML-mTIM-1/Fc and those treated with control IgG2a antibody is shown in Figure 3. The incidence of EAE in the control group was significantly (p < 0.02, unpaired t-test) higher than that observed in the ML-mTIM-1/Fc treated group. The days of onset was also significantly (p < 0.006) delayed in the mice treated with ML-mTIM-1/Fc (Figure 4) in comparison to those in the control group. The mean (± SD) days of onset in the control and ML-mTIM-1/Fc treated groups were found to be 11 ±1 and 13 ±2, respectively. Similarly, the maximum scores attained by the mice in the control group were also significantly (p < 0.0001) higher than those in the ML-mTIM-1/Fc treated groups (Figure 5). The mean disease progression over time is given in Figure 6.

[00108] These results show that ML-mTIM-1/Fc lowers the incidence (Figure 3), significantly delays the onset of disease (p<0.006, Figure 4) and suppresses the severity of disease (p<0.0001, Figure 5) in a model of relapsing-remitting EAE in SJL/J mice. These results clearly demonstrate that administration of ML-mTIM-1/Fc fusion protein curtails the acute phase of relapsing remitting EAE.

EXAMPLE V

TIM-I Fusion Protein (TIM-l/Fc) Reduces Hair Loss in a Murine Model of Alopecia

Areata

[00109] This example describes the effect of TIM-l/Fc on reducing hair loss in a murine model of alopecia areata (AA).

[00110] For full thickness skin graft and treatment, alopecic skin from eighteen

C3H/HeJ mice who had developed AA was grafted onto the back of unaffected mice according to the technique previously described (McElwee et al., J. Invest. Dermatol. 111 :797-803 (1998)). 100 μg TIM-l/Fc or IgG control (mouse IgG2a, R and D Systems, Minneapolis, Minn., #MAB003) was given intraperitoneally (IP) at the onset of alopecia starting at week five, as shown in Figure 16. Hair loss was examined weekly, and photographs taken. The extent of hair loss was quantified by image analysis software (Sigma Scan, Jandel Scientific, San Rafael, CA) based on photographs taken. Briefly, areas of alopecic skin, with the exception of the graft, were outlined using the software. The total area of alopecic skin was calculated for both the ventral and dorsal surface and was converted to a percentage of total skin surface area visible in the photo.

[00111] To generate TIM-l/Fc fusion protein, mouse TIM-l/Fc fusion protein was expressed from a shuttle plasmid vector. The basic vector carries bacterial and eukaryotic resistance genes as well as a multiple cloning site flanked by an intron A-containing CMV enhancer/early promoter element, a human CD5 leader sequence, and a classic bovine growth hormone (BGH) polyA site. A mouse non-lytic IgG2a/Fc (Zheng et al., .L Immunol. 154:5590-5600 (1995)) was amplified by PCR from cDNA containing the mouse non-lytic IgG2a/Fc and cloned into the expression vector.

[00112] Subsequently, the extra-cellular immunoglobulin (Ig) domain of the mature mouse TIM-I gene (Mclntire et al. , Nat. Immunol. 2: 1109-1116 (2001)) was inserted into the plasmid vector in frame with and 5' of the non-lytic mIgG2a Fc region to yield the final expression vector. The accuracy of the plasmid construct was confirmed by DNA sequencing. [00113] 293 cells were transiently transfected with an endotoxin-free preparation of the mTIM-1/Fc expression vector and used to produce mouse TIM-I /Fc fusion protein. Briefly, culture media were collected, clarified by centrifugation, concentrated, and the secreted TIM-I /Fc was immobilized via Protein G. The column was washed and TIM- 1/Fc fusion protein eluted by low pH (pH=2.75). Fractions were collected in tubes containing neutralization buffer (IM Tris-HCL, pH 9.0). As necessary, the eluted TIM- 1/Fc proteins were further purified by size exclusion chromatography to remove aggregates. Purified protein was dialyzed against PBS, sterile filtered, and stored in aliquots at -8O⁰C until use.

[00114] To analyze lymphocyte proliferation and cytokine production, lymph node cells were analyzed after two additional injections of TIM-l/Fc or control IgG for in vitro lymphocyte proliferation and cytokine production. Briefly, skin-draining lymph nodes were collected, and single cell suspensions were made. Cells (2 x 10⁵ cells/well in 96- well flat-bottom plates) were cultured in RPMI medium with 10% fetal calf serum in the presence of medium alone, and with various concentrations of Concanavalin A (Con A) (Amersham, Piscataway, NJ). After red blood cell (RBC) lysis with ACK lysing solution (Invitrogen, Carlsbad, CA), the cells were washed and re-suspended in complete media (RPMI 1640, 10% FBS, GlutaMAX™, 5μM, β-mercaptoethanol), and adjusted to 5xlO⁶ viable cells/ml. Cells (lOOμl per well) were incubated in quadruplicate with increasing amounts of Concanavalin A (Con A) in a final volume of 200 μl in flat-bottom, opaque white-wall plates for 96 hours at 37⁰C and 5% CO₂. Sixteen hours prior to harvest, the cells were pulsed with 10 μM bromdeoxyuridine (BrdU) and processed according to the procedures for the Delfia Proliferation Assay (Perkin-Elmer, Wellesley, MA). Anti-BrdU Europium-based fluorescence was detected using a Wallac-1420 Victor-2 time-resolved fluorimeter. Results are represented as relative fluorescence units (RFU) ± standard error of the mean (SEM). After 12O h of culture, supernatants were collected for cytokine analysis. Levels of cytokines IL-4, IL-5, IL-9, IL-13, IFN-γ, IL-2, and TNF-alpha in culture supernatants were determined by Cytometric Bead Array (Becton Dickenson Biosciences, San Jose, California), according to the manufacturer's protocols.

[00115] For statistical analysis, student's t test was used to compare treatment groups. P-values of < 0.05 were considered significant. [00116] Alopecia developed in all the recipient animals by week five after the full thickness skin graft from affected animals. As shown in Figure 17, mice that received TIM-l/Fc had a significantly smaller surface area of alopecia compared to Ig control group (p=<0.05). In vitro immune response from the draining lymph nodes was examined. As shown in Figure 18, significantly stronger lymphocyte proliferation to Con A was observed in the TIM-I/ Fc treated group compared to the IgG control group. Furthermore, as shown in Figure 19, an augmentation of Th2 type cytokines in response to Con A stimulation was observed. This included enhanced IL-4 (P<0.05) and IL-9 (PO.05), with a trend toward significant increases for IL-5, IL-IO, and IL- 13 in the TIM- l/Fc treated group compared to the Ig control group. However, TIM-I/ Fc had no significant effect on ThI cytokines such as interferon gamma, TNF-alpha, and IL-2 compared to the IgG control group.

[00117] The observed in vivo effects of TIM-l/Fc administration were accompanied by in vitro immunologic effects. Most notably, enhanced lymphocyte proliferation was observed in vitro. The fact that modulation of immunity was observed with Con A stimulation, a potent mitogen, further substantiates the important role the TIM family plays in modulation of the immune response. Overall, an increase in Th2 cytokines was observed in the model of alopecia areata. However, a concomitant decrease in ThI cytokines was not observed. Nevertheless, this augmentation of Th2 cytokines, and perhaps other immunologic events, correlated with a diminution of alopecia in the TIM- l/Fc treated animals.

[00118] These results are the first to suggest in vivo effects of TIM-l/Fc in an alopecia areata mouse model. Thus, TIM-l/Fc offers a novel approach to treating alopecia areata and other autoimmune diseases.

EXAMPLE VI

Efficacy of Dimerized Mucinless TIM-l/Fc on Disease Course of Experimental Autoimmune Encephalomyelitis (EAE)

[00119] This example describes the effects of anti-TIM-4 on the course of relapsing-remitting EAE in SJL/J mice.

[00120] The materials and methods and reagents used were as follows. The rat anti-mouse TIM-4 antibody was prepared as described in Example III. The reagents used were as described in Example IV. Six to eight weeks old female SJL/J mice were purchased from Jackson Laboratories (Bar Harbor ME). The EAE induction and anti- TIM-4 administration schedule is shown in Figure 20. Briefly, on day 0, mice were immunized with 200 μg Of PLPi_39-151 peptide emulsified in CFA (Mycobacterium tuberculosis concentration at 200 μg) in the right and left flanks. Following the injection of the PLPi₃₉-i₅i peptide in CFA, 100 ng of PT was injected intravenously via the tail vein. The animals were then monitored daily for disease symptoms. The disease was scored as follows: 0: no abnormality, healthy; 1 : limp tail or hind limb weakness; 2: limp tail and hind limb weakness; 3: limp tail and partial paralysis of hind or fore limbs; 4: limp tail and complete hind or fore limb paralysis or quadriplegia; and 5: death.

[00121] For administration of the proteins, TIM-4 and control antibodies were administered. Starting from day 8, mice were injected with 100 μg/dose of anti-TIM-4 or IgG2a intraperitoneally. A total of 3 injections at 48 hr intervals were administered and disease progression monitored.

[00122] The incidence of EAE in mice treated with anti-TIM-4 and those treated with control IgG2a antibody is shown in Figure 21. The incidence of EAE in the control group was significantly higher than that observed in the anti-TIM-4 treated group. The mean disease progression over time is given in Figure 22.

[00123] These results show that anti-TIM-4 lowers the incidence (Figure 21), and suppresses the severity of disease (Figure 22) in a model of relapsing-remitting EAE in SJL/J mice. These results clearly demonstrate that administration of anti-TIM-4 curtails the acute phase of relapsing remitting EAE.

[00124] Throughout this application various publications have been referenced.

The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims

What is claimed is:

1. A method for ameliorating a sign or symptom associated with an autoimmune disease, comprising administering a TIM-I ligand targeting molecule to a subject, wherein said TIM-I ligand targeting molecule is dimeric.

2. The method of claim 1, wherein said autoimmune disease is multiple sclerosis.

3. The method of claim 1, wherein said autoimmune disease is alopecia areata.

4. The method of claim 1, wherein said autoimmune disease is selected from rheumatoid arthritis, autoimmune diabetes mellitus, systemic lupus erythematosus, autoimmune lymphoproliferative syndrome (ALPS), and inflammatory bowel disease.

5. The method of claim 1, wherein said TIM-I ligand targeting molecule is a TIM-4 antibody.

6. The method of claim 5, wherein said TIM-4 antibody is target-cell depleting.

7. The method of claim 5, wherein said TIM-4 antibody is non target-cell depleting.

8. The method of claim 1, wherein said TIM-I ligand targeting molecule is a TIM-I-Fc fusion polypeptide.

9. The method of claim 8, wherein the Fc portion of said TIM-I-Fc fusion polypeptide is target-cell depleting.

10. The method of claim 8, wherein the Fc portion of said TIM-I-Fc fusion polypeptide is non target-cell depleting.

11. A method for ameliorating a sign or symptom associated with transplant rejection, comprising administering a TIM-I ligand targeting molecule to a subject, wherein said TIM-I ligand targeting molecule is dimeric.

12. The method of claim 11, wherein said TIM-I ligand targeting molecule is a TIM-4 antibody.

13. The method of claim 12, wherein the TIM-4 antibody is target-cell depleting.

14. The method of claim 12, wherein the TIM-4 antibody is non target-cell depleting.

15. The method of claim 11, wherein said TIM-I ligand targeting molecule is a TIM-I-Fc fusion polypeptide.

16. The method of claim 15, wherein the Fc portion of said TIM-I-Fc fusion polypeptide is target-cell depleting.

17. The method of claim 15, wherein the Fc portion of said TIM-I-Fc fusion polypeptide is non target-cell depleting.

18. A fusion polypeptide comprising a TIM-I polypeptide fragment containing the TIM-I IgG fragment fused to a Fc.

19. A pharmaceutical composition, comprising the fusion polypeptide of claim 14 in a pharmaceutical carrier.

20. A pharmaceutical composition, comprising a TIM-4 antibody in a pharmaceutical carrier.