MX2007010738A - Kim-1 antibodies for treatment of th2-mediated conditions - Google Patents

Abstract

Compositions and methods for treating Th2- and ThI -mediated disease are provided.

Description

ANTIBODIES KIM-1 FOR THE TREATMENT OF CONDITIONS CAUSED BY TH2 BACKGROUND OF THE INVENTION It is believed that atopic diseases such as allergic asthma and atopic dermatitis involve a pathogenic change to the predominant immunity of Th2 (Umetsu et al., 2002, Nat Immunol 3: 715-20.In the onset of asthma, the production of the Th2 cytokine leads to the influx of eosinophils in the lung, the activation of eosinophils, the production of IgE and the activation and degranulation of mast cells produced by IgE, and the accumulation of mononuclear cells in the pulmonary interstitial space, where the lymphocytes T and activated granulocytes continue to secrete Th2 cytokines, chemokines, and effector molecules, thereby encouraging lung inflammation to continue.The TAPR locus containing KIM gene family has been implicated in the development of atopic inflammation in mice, and allelic variation of KIM-1 has been associated with the incidence of atopy in the analyzes in patient populations (Mclntire et al., 2001, Nat Immunol 2: 1109-16; Mclntire et al., 2003, Nature 425: 576).

BRIEF DESCRIPTION OF THE INVENTION The invention is based, at least in part, on the discovery that agents, such as antibodies, that bind particular regions of KIM-1, can differentially modulate the immunity produced by Thl and / or Th2. . For example, agents that bind the stem region of KIM-1 or the sialic acid binding regions of KIM-1 can modulate the expression of Th2 cytokines and can be used to treat a Th2 disorder, for example asthma.; and the agents that bind the particular epitopes within the mucin region of KIM-1 can reduce a pathogenic Thl response and can be used to treat a disorder produced by Thl, for example, inflammatory disorders or autoimmune disorders such as inflammatory disease. of the intestine (IBD), Crohn's disease, multiple sclerosis, diabetes, rheumatoid arthritis, psoriasis, acute graft-versus-host disease (GVHD), transplant, pancreatitis, delayed-type hypersensitivity (DTH). The compositions and methods useful in the treatment of disorders produced by Th2 and Thl are provided.

In one aspect, the invention provides methods for treating conditions caused by Th2, for example, asthma (in particular allergic asthma), allergic rhinitis, allergy, eczema, and other atopic conditions. The methods include administering to a mammal, preferably a human, having a condition elicited by Th2, an agent that binds the stem region of KIM-1 or the binding motive with sialic acid of KIM-1. For example, the method may include administering a pharmaceutical composition containing a monospecific antibody, for example, a monoclonal antibody. (or antigen binding fragment thereof) that binds the stem region of KIM-1 or the binding motive with sialic acid of KIM-1, in an amount and for a sufficient time to treat the condition. The stem region of KIM-1 is identified herein as a charged domain containing the relatively conserved N-linked glycosylation sites present between the mucin domain and the transmembrane domain of KIM-1. The stem region of human KIM-1 and the binding motif with sialic acid are shown in Fig. 1. It should be understood that the terminal N and C of these regions as defined herein are approximate and can contain some (eg, 1, 2 or 3) more or less contiguous residues of the KIM-1 sequence. In one embodiment, the agent is an antibody that binds to the stem region of human KIM-1. For example, the antibody binds a peptide having the sequence DGNDTVTESSDGL NNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1). In one embodiment, the antibody binds to a peptide having the sequence LLTANTTKG (amino acids 262-270 of SEQ ID NO: 1), HSLLTANTTKG (amino acids 260-269 of SEQ ID NO: 1), FLEHSLLTANTTKG (amino acids 257-270 of SEQ ID NO: l) or NQTQLFLEHSLLTANTTKG (amino acids 252-270 of SEQ ID NO: 1). In other embodiments, the antibody binds to a peptide having the amino acid sequence 236-250 or 236-258 of SEQ ID NO: 1. In one embodiment, the antibody binds to the binding motif with sialic acid of KIM- 1. For example, the antibody binds, at least in part, to an epitope contained within or overlap is a peptide having the sequence GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1). In one embodiment, the antibody binds to a reduced and non-reduced protein, i.e., binds to a linear epitope of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid residues of GVYCCRVEHRG FNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1). In another embodiment, the antibody binds to a structural epitope to which one or both of the GVYCCRVEHRG FNDMKITVSLEIVPP sequences (amino acids 81-107 of SEQ ID NO: 1) and RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1) contribute. ). In one embodiment, the epitope can be a structural epitope contained in the human KIM-1 sequence corresponding to a TPCK fragment of 8 kDa trypsin from the IgGl Fe fusion of human IgV from the recombinant mouse KIM-1. In one embodiment, the antibody interferes with one or more of the residues R86, W92, and F93 of SEQ ID NO: 1, which are required for binding with sialic acid. While it should be understood that the methods described herein are not limited by any particular mechanism or theory, the antibody may have one or more of the following characteristics: (a) interferes with an interaction of the binding motif with sialic acid in the IgV domain of KIM-1 with the carbohydrates exhibited at one or more sites of N-glycosylation of the stem region of KIM-1, (b) binds or hinders sterically one or more N-linked glycosylation sites in the stem region, (c) inhibits the down-regulation of KIM signaling -1, (d) is an agonist antibody, for example, when it binds it stimulates or increases the signaling in the 3 'direction with KIM-1, (e) it blocks the multimerization of KIM-1, (f) binds or hinders sterically the interaction of the stem region with a co-receptor or ligand to interrupt normal function, (g) interfere with an interaction of the binding motive with sialic acid in the IgV domain of KIM-1 with the carbohydrates exhibited in one or more O-glycosylation sites of the mucin region adjacent to the stem region of KIM-1, (h) alters the structural characteristics of the Ig domain to change the protein conformation, for example, by altering or interfering with the disulfide bond or hydrogen, (i) alters the characteristics is of the KIM-1 Ig domain to change the binding to other proteins such as the KIM-1 ligand. In one embodiment, the antibody does not inhibit KIM-1 spillage from the cell surface, by example, it does not inhibit the spill of KIM-1 from Ebna 293 (E293) cells in culture. In preferred embodiments the antibody is monospecific, for example, the antibody is a monoclonal antibody, for example, a humanized or fully human monoclonal antibody or an antigen binding fragment. In one modality, the condition is allergic asthma. In this embodiment, the method also optionally includes identifying a subject who is at risk, or has, allergic asthma. Optionally, the method also includes evaluating a symptom of asthma in the subject, e.g., IgE levels, airway hypersensitivity, cough, wheezing, chest tightness, dyspnea, contraction of smooth muscle in the respiratory tract, bronchial secretion of mucus, inflammation, vasodilation, collection of inflammatory cells (for example, neutrophils, monocytes, macrophages, lymphocytes, eosinophils), goblet cell hyperplasia, inflammatory mediator release by mast cells or migrating inflammatory cells. The evaluation step can be carried out before, during and / or after the administration step. The evaluation can be done by a doctor, or another person who is in the care of the health or by the subject. The evaluation may be performed one or more times, for example, one or more times after administration, for example, at least twice during a period of one week, one month, two months, three months, six months after the administration. administration, or more time. In a preferred embodiment, the method includes determining whether the administration of the agent (or multiple administrations) reduces or reduces the severity or initiates one or more of the symptoms of a respiratory tract disease in the subject. In some modalities, the antibody is coadministered with a second agent effective to treat asthma in the subject, eg, a corticosteroid, bronchodilator, a leukotriene modifier, anti-inflammatory agent, an anti-IgE agent (eg, an anti-IgE antibody) , for example, omalizumab (Xolair®)). "Co-administered" or "administered in combination" means administration at the same time or within a range, for example, a week, such that the effects of the substances on the patient overlap.

In another embodiment, the condition is allergy, for example, food allergy or temporary allergy (for example, by pollen). An allergy diagnosis can be made by one or more of: the administration of a skin test with allergens; the determination of serum IgE concentration (for example, IgE> 300 ng / ml); and the determination of allergen-specific IgE or IgG antibodies in serum. The antibody can be administered in one or more of the following periods: before atopic exposure of a subject to the allergen; after exposure to the allergen even before the onset of symptoms; at the time of onset of symptoms; after the onset of symptoms. In one embodiment, the agent is administered as a treatment course, for example, in periodic administrations of predetermined frequency, for example, daily, weekly, twice a week or monthly. In some embodiments, an antibody may be administered for a period of time and / or in an amount sufficient to reduce (e.g., substantially reduce) the frequency or severity of wheezing, cough, failure. respiratory, or tightness in the chest, for example, over a period of time, for example, 3 months, 6 months, a year or more. In another aspect, the invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds to the stem region of KIM-1. The antibody does not inhibit KIM-1 efflux of E293 cells in the culture. In one embodiment, the antibody binds to the stem region of human KIM-1. For example, the antibody binds to a peptide having the sequence DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1). In one embodiment, the antibody binds to a peptide having the sequence LLTANTTKG (amino acids 262-270 of SEQ ID NO: 1), HSLLTANTTKG (amino acids 260-270 of SEQ ID NO: 1), FLEHSLLTANTTKG (amino acids 257- 270 of SEQ ID N0: 1) or NQTQLFLEHSLLTANTTKG (amino acids 252-270 of SEQ ID NO: 1). In other embodiments, the antibody binds to a peptide having the amino acid sequence 241-254, 242-258, 242-255 of SEQ ID NO: 1. In another aspect, the invention provides an isolated antibody, or the fragment of binding with antigens thereof that binds specifically to a reason for binding with sialic acid of KIM-1. For example, the antibody binds, at least in part, to an epitope contained within or overlapping with the peptide having the sequence GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) or RGSCSLFTCQNGIV (amino acids 29-42). of SEQ ID NO: 1). In one embodiment, the antibody binds to reduced and unreduced protein, i.e., binds to a linear epitope of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid residues of GVYCCRVEHRG FNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID N0: 1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1). In another embodiment, the antibody binds to a structural epitope to which one or both of the sequences GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) and RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1) contribute. In some embodiments, the epitope is a structural epitope contained in a region of human KIM-1 that corresponds to a TPCK fragment of 8 kDa trypsin from the IgG-human IgGl Fe fusion of recombinant mouse KIM-1. In one embodiment, the antibody interferes with one or more of the residues R86, W92, and F93 of SEQ ID NO: 1, which are required for binding with sialic acid.

The isolated antibody can have one or more of the following characteristics: (a) it interferes with an interaction of the binding motif with sialic acid in the IgV domain of KIM-1 with the carbohydrates exhibited at one or more sites of N-glycosylation of the Stem region of KIM-1, (b) sterically binds or hinders one or both N-linked glycosylation sites in the stem region, (c) inhibits the down-regulation of KIM-1 signaling, (d) is an agonist antibody, for example, with binding stimulates or enhances signaling in the 3 'direction with KIM-1, (e) blocks the KIM-1 multimerization, (f) binds or hinders sterically the interaction of the stem region with a co-receptor or ligand to interrupt normal function, (g) interferes with an interaction of the binding motive with sialic acid in the IgV domain of KIM-1 with the carbohydrates exhibited at one or more O-glycosylation sites of the mucin region adjacent to the stem region of KIM-1, (h) alters the structural characteristics of the Ig domain to change the protein conformation , for example, by altering or interfering with disulfide or hydrogen bonding, (i) alters the structural or functional characteristics of the KTM-1 Ig domain to change the binding to other proteins such as the KIM-1 ligand. In another aspect, the invention incorporates a method for treating a condition caused by Thl, for example, a condition characterized by an increased pathogenic response or Thl. These conditions include inflammatory and / or autoimmune disorders such as inflammatory bowel disease (IBD), Crohn's disease, multiple sclerosis, diabetes, rheumatoid arthritis, psoriasis, acute graft-versus-host disease (GVHD), transplantation, pancreatitis, delayed-type hypersensitivity. (DTH). The method includes administering to a mammal, preferably a human being, having a condition produced by Thl, an agent, for example, an antibody, that binds to an epitope contained in the sequence VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT (residues 200-235 of SEQ ID NO: 1). For example, the method may include administering a pharmaceutical composition containing a monospecific antibody, for example, a monoclonal antibody. (or antigen binding fragment) that binds the specific region of KIM-1, in an amount and for a sufficient time to trait the condition. The specific region of KIM-1 was found in a alternately assembled variant of KIM-1 in the mouse. It should be understood that the terms N and C of this region as defined herein are approximate and may contain some (eg, 1 or 2) plus or minus contiguous residues of the KIM-1 sequence. In one embodiment, the antibody binds to the reduced and unreduced protein. In preferred embodiments, the antibody is monospecific, e.g., the antibody is a monoclonal antibody, e.g., a humanized or fully human monoclonal antibody or an antigen binding fragment. In one modality, the condition is inflammatory bowel disease (IBD), Chron, rheumatoid arthritis, psoriasis, acute graft-versus-host disease (GVHD), transplant, pancreatitis, or delayed-type hypersensitivity (DTH). The method also optionally includes identifying a subject who is at risk, or who has, any of the conditions listed. In a preferred embodiment, the method includes determining whether the administration of the agent (or multiple administrations) reduced or not the severity or beginning of one or more symptoms of the condition of the subject. In some embodiments, the antibody is coadministered with a second agent effective to treat the condition in the subject, for example, a corticosteroid or other anti-inflammatory agent, DMARD, anti-TNF therapy or anti-CD20 therapy. "Co-administered" or "administered in combination" means administration at the same time or within a range, for example, a week, such that the effects of the substances on the patient overlap. In one embodiment, the agent is administered as a course of treatment, for example, in periodic administrations of predetermined frequency, for example, daily, weekly, twice a week or monthly. In some embodiments, an antibody may be administered for a period of time and / or in an amount sufficient to reduce (e.g., substantially reduce) the frequency or severity of symptoms, e.g., over a period of time, e.g. months, 6 months, a year or more. In the sense in which it is used in the present, the terms "treat," "treating," and "treatment" refers to the administration of a therapy in an amount, manner, and / or effective manner to ameliorate or diminish a symptom or parameter that is characterized by a pathological condition; to reduce the severity of a symptom or a parameter that is characterized by a pathological condition; to prevent, diminish or reverse the progression of the pathological condition; or to prevent one or more of the symptoms or parameters of the pathological condition. As used herein, an "agent that binds" to a particular domain of KDVI-l refers to any compound that binds to the specific domain with a Kd less than 10"6 M. An example of a binding agent with KIM-1 is a binding protein with KIM-1, for example, a binding antibody with KIM-1, preferably a monospecific antibody. In the sense in which the terms "binding region with sialic acid", "binding motive with sialic acid", and "required for binding with sialic acid" are used herein, and variants of those terms refer to amino acid residues, amino acid sequences, and secondary or tertiary structures of amino acids that are similar or homologous to those amino acid residues, amino acid sequences, and secondary and tertiary structures of amino acids identified in the Ig-like lectin family of sialic acid binding (Siglecs) that are required for carbohydrate binding. The term "antibody binding fragment with antigens thereof" encompasses proteins that include at least one variable region of immunoglobulin, for example, an amino acid sequence that provides an immunoglobulin variable domain or an immunoglobulin variable domain sequence sufficient to specifically bind an antigen. For example, the term includes an antigen binding protein having a heavy chain variable region (H) (abbreviated herein as VH), and a light chain (L) variable region (abbreviated herein as VL) . In another example, the term includes an antigen-binding protein that includes two heavy chain variable regions (H) and two light chain (L) variable regions. The term encompasses the binding fragments with antibody antigens (e.g., single chain antibodies, Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, and fragments dAb) as well as complete antibodies, for example, intact immunoglobulins of the types IgA, IgG, igE, IgD, igM (as well as subtypes thereof). The immunoglobulin light chains can be of the kappa or lambda types. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and / or cytotoxicity produced by supplements, or it can be non-functional for one or both of these activities. The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "raster regions" (FR). The degree of FRs and CDRs has been defined exactly (see, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). The definitions of Kabat are used here. Each VH and VL is typically composed of these three CDRs and four FRs, arranged from the amino terminus to the term carboxyl in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The above summary and the following description are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is an annotated polypeptide sequence (SEQ ID NO: 1) of human KIM-1 (without the signal sequence and without the MTTVP insertional polymorphism) showing the various domains described herein. Fig. 2 is a graph showing the percentage of eosinophils (and axis) present in the bronchial lavage fluid (BAL) after aerosol inoculation with OVA and treatment with 3A2. Fig. 3 is a graph showing the proliferation of drained (bronchial) lymph node cells after ex vivo staging with the OVA antigen and treatment with 3A2. The Y axis is treated with tritium and incorporation of thymidine in cpm. FIG. 4 is a graph showing the drained (bronchial) response of lymph node cells for ex vivo stimulation with the OVA antigen. LNs were collected after aerosol inoculation with OVA, and then cultured with OVA ex vi ve and treatment with 3A2. Supernatants were extracted from these cultures and analyzed for the production of the TH2 cytokine (IL-4, IL-5 and IL-10). The Y axis shows picagrams / ml. Fig. 5 shows the binding curves generated for the interaction of 3A2 with the immobilized proteins. MAb 3A2 bound equivalently to mKIM-1-ECD-Fc (circles), to mKIM-1-137-216-Fc (triangles), and to mKIM-1 - 196-216-Fe (diamonds), although they could not bind to mKIM-1-IgV-Fc (squares). Fig. 6 is a graph showing the percentage of eosinophils and lymphocytes (y-axes) present in the bronchial lavage fluid (BAL) after aerosol inoculation with OVA and treatment with 4A2. Fig. 7 is a graph showing the drained (bronchial) response of lymph node cells for ex vivo stimulation with the OVA antigen and treatment with 4A2. The LNs were collected after the aerosol inoculation with OVA, and then they were grown with OVA ex vi vo.

Supernatants were extracted from these cultures and analyzed for the production of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13). The Y axis shows picagrams / ml. Fig. 8 is a graph showing the percentage of eosinophils (y axis) present in the bronchial lavage fluid (BAL) after aerosol inoculation with OVA and therapeutic treatment with 4A2. Fig. 9 shows the binding curve of the mAb 4A2 to the purified proteins of KIM-1.

DETAILED DESCRIPTION OF THE INVENTION As described herein, the targeted regions of KIM-1 with antibody therapy exert critical control over the expression of the Th2 and Th1 cytokines and provide therapeutic strategies for the treatment of diseases caused by Th2 and Th2. other atopic disorders, and for diseases caused by Thl.

Generation of antibodies The antibodies described herein (for example, antibodies that bind to the stem region of KIM-1 or to the binding motif with sialic acid of KIM-1) can be generated by immunization, for example, using an animal, or by in vitro methods such as phage display. A polypeptide that includes the white epitope of KIM-1 (e.g., the stem region of KIM-1 or the sialic acid binding motif of KIM-1) can be used as an immunogen. In other embodiments, a larger portion of the KIM-1 polypeptide, such as the extracellular domain, can be used as an immunogen and the resulting antibodies can be selected for reactivity to the desired KIM-1 region or domain. In one embodiment, the immunized animal contains immunoglobulin-producing cells with natural, human, or partially human immunoglobulin loci. In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, it is possible to engineer the strains of mice with deficiency for the production of mouse antibodies with large fragments of human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity can be produced and selected. See, for example, XenoMouseMR, Green et al. Nature Genetics 7: 13-21 (1994), US 2003-0070185, U.S. Patent No. 5,789,650, and WO 96/34096. Non-human antibodies can also be produced for KIM-1, for example, in a rodent. The non-human antibody can be humanized, for example, as described in U.S. Patent No. 6,602,503, EP 239,400, U.S. Patent No. 5,693,761, and U.S. Patent No. 6,407,213. EP 239 400 (Winter et al.) Describes the alteration of antibodies by substitution (within a given variable region) of their complementarity determining regions (CDRs) for one species with those of another. The CDR-substituted antibodies may be less likely to produce an immune response in humans compared to true chimeric antibodies because the CDR-substituted antibodies contain considerably less non-human components. (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Typically, the CDRs of a murine antibody substituted in the corresponding regions in a human antibody using technology with recombinant nucleic acid to produce the sequences encoding the desired substituted antibody. Gene segments of the human constant region of the desired isotope (in general gamma I for CH and kappa for CL) can be added and the humanized heavy and light chain genes can be co-expressed in mammalian cells to produce the soluble humanized antibody. Queen et al., 1989 and WO 90/07861 have described a process that includes selecting human V frame regions by computerized analysis for the optimal homology of protein sequences to the V region frame of the original murine antibody, and modeling the structure tertiary of the murine V region to visualize the amino acid residues of the plot that probably interact with murine CDRs. These murine amino acid residues are then superimposed on the homologous human frame. See also United States Patents Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101. Tempest et al., 1991, Biotechnology 9, 266-271, use, as a standard, the V region frames derived from the heavy and light chains of NEWM and REI, respectively, for the CDR graft without the radical introduction of waste from mouse. An advantage of using the procedure of Tempest et al. to form the humanized antibodies based on NEWM and REI is that the similar three-dimensional structures of the NEWM and REI variable regions are known to come from X-ray crystallography and in this way the specific interactions between the frame residues can be modeled of the CDR and V region. The non-human antibodies can be modified to include substitutions that insert the human immunoglobulin sequences, for example, consensual human amino acid residues at particular positions, for example, into one or more (preferably at least five, ten, twelve, or all) the following positions: (in the FR of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and / or (in the FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H , 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and / or 103H (according to the Kabat numbering). See, for example, U.S. Patent No. 6,407,213.

Fully human monoclonal antibodies that bind to the desired regions of KIM-1 can be produced, for example, by using human splenocytes primed in vi tro, as described by Boerner et al., 1991, J. Immunol. , 147, 86-95. They can be prepared by cloning repertoires as described by Persson et al., 1991, Proc. Nat. Acad. Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol. Methods 141, 227-236; also U.S. Patent No. 5,798,230. Large libraries for displaying non-immunized phage can also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (see, eg, Vaughan et al, 1996; Hoogenboom et al., (1998) Immunotechnology. 4: 1-20; and Hoogenboom et al. (2000) Immunol Today 2: 371-8; US 2003-0232333). As used herein, "an immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the similar structure of an immunoglobulin variable domain. For example, the sequence may include all or a portion of the amino acid sequence of a variable domain that occurs in nature. For example, the sequence may omit one, two or more terminal N or C amino acids, the internal amino acids may include one or more additional terminal amino acids or insertions, or may include other alterations. In one embodiment, a polypeptide that includes an immunoglobulin variable domain sequence can be associated with another immunoglobulin variable domain sequence to form a white binding structure (or an "antigen binding site"), eg, a structure that interacts with a specific region of KIM-1. The V H or V L chain of the antibody may also include all or a portion of a heavy or light chain constant region, to form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two immunoglobulin light chains. The heavy and light chains of immunoglobulin can be connected by disulfide bonds. The heavy chain constant region typically includes three constant domains, CH1, CH2 and CH3. The light chain constant region typically includes a CL domain. The region The heavy and light chain variable contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically produce the binding of the antibody to tissues or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. One or more regions of an antibody can be human, effectively human, or humanized. For example, one or more of the variable regions can be human or indeed human. For example, one or more of the CDRs, for example, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3, can be human. Each of the light chain CDRs can be human. HC CDR3 can be human. One or more of the raster regions can be human, for example, FR1, FR2, FR3, and FR4 of the HC or LC. In one embodiment, all frame regions are human, e.g., derived from a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, human sequences are germline sequences, for example, encoded by a germline nucleic acid. One or more of the Constant regions can be human, effectively human, or humanized. In another embodiment, at least 70, 75, 80, 85, 90, 92, 95, or 98% of the raster regions (e.g., FR1, FR2, and FR3, collectively, or FR1, FR2, FR3, and FR4 , collectively) or the total antibody can be human, effectively human, or humanized. For example, FR1, FR2, and FR3 can collectively be at least 70, 75, 80, 85, 90, 92, 95, 98, or 99% identical to a human sequence encoded by a human germline segment. A variable region of "effectively human" immunoglobulin is a variable region of immunoglobulin that includes a sufficient number of amino acid positions in the human framework such that the variable region of immunoglobulin does not produce an immunogenic response in a normal human being. An "effectively human" antibody is an antibody that includes a sufficient number of human amino acid positions in such a way that the antibody does not produce an immunogenic response in a normal human being. A "humanized" variable region of immunoglobulin is a variable region of immunoglobulin that is modified so that the modified produces less than an immune response in a human being than the unmodified form does, for example, is modified to include a sufficient number of amino acid positions in the human framework such that the immunoglobulin variable region does not produce an immunogenic response in a normal human being. Descriptions of "humanized" immunoglobulins include, for example, U.S. Patent No. 6,407,213 and U.S. Patent No. 5,693,762. In some cases, the humanized immunoglobulins may include a non-human amino acid at one or more positions of the frame amino acids. The whole or a portion of an antibody can be encoded by an immunoglobulin gene or a segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgG1, IgG2, IgG3, IgG3, IgG4), delta, epsilon and mu constant genes, as well as the countless region genes Immunoglobulin variable. The "light chains" of full length immunoglobulin (approximately 25 Kd or 214 amino acids) are encoded by a variable region gene in the NH2 term (approximately 110 amino acids) and a kappa or lambda constant region gene in the COOH term. The "heavy chains" of full-length immunoglobulin (approximately 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (approximately 116 amino acids) and one of the other aforementioned constant region genes, for example, gamma (which encodes approximately 330 amino acids). The term "antigen binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retains the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab ') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge in the axis region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single branch of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544- 546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. In addition, although the two domains of the Fv, VL and VH fragment are encoded for the separated genes, they can be linked, using recombinant methods, by means of a synthetic linker that allows them to be constituted as a single protein chain in which the regions VL and VH form pairs to form monovalent molecules known as individual chain Fv (scFv). See, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nati Acad. Sci. USA 85: 5879-5883.

Production of antibodies Antibodies can be produced in prokaryotic and eukaryotic cells. In one embodiment, antibodies (e.g., scFv's) are expressed in a yeast cell such as Pichia (see, e.g., Powers et al (2001) J Immunol. Methods 251: 123-35), Hanseula, or Saccharomyces. . In one embodiment, antibodies, particularly full-length antibodies, eg, IgG, are produced in mammalian cells.

Exemplary host mammalian cells for recombinant expression include Chinese Hamster Ovary (CHO cells) (which includes dhfr-CHO cells, described in Urlaub and Chasin (1980) Proc. Nati. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601-621), lymphocytic cell lines, eg, NSO myeloma cells and SP2 cells, COS cells, K562, and a cell from a transgenic animal, for example, a transgenic mammal. For example, the cell is a mammary epithelial cell. In addition to the nucleic acid sequence encoding the immunoglobulin domain, recombinant expression vectors can carry additional acidic nucleic acid sequences, such as sequences that regulate vector replication in host cells (eg, the origins of replication). ) and the selectable marker genes. The selectable marker gene facilitates selection of the host cells into which the vector has been introduced (see, for example, U.S. Patent No. 4,399,216, 4,634,665 and 5,179,017). Exemplary selectable marker genes include the gene of dihydrofolate reductase (DHFR) (for use in dhfr host cells - with selection / amplification of methotrexate) and the neo gene (for selection G418). In an exemplary system for the recombinant expression of an antibody (e.g., a full-length antibody or an antigen-binding portion thereof), a recombinant expression vector encoding both the heavy chain of the antibody and the light chain of the antibody in dhfr-CHO cells by transfection supplied by calcium phosphate. Within the recombinant expression vector, the heavy and light chain genes of the antibody are each operably linked to enhancer / promoter regulatory elements (e.g., derivatives of SV40, CMV, adenovirus and the like, such as a regulatory enhancer element). CMV / AdMLP promoter or an SV4 O enhancer regulatory element / AdMLP promoter) to drive high levels of transcription of the genes. The vector of the recombinant expression also carries a DHFR gene, which allows the selection of CHO cells that have been transfected with the vector when using the selection / amplification of methotrexate. The cells Selected transformant hosts are cultured to allow expression of the heavy and light chains of the antibody and the intact antibody is recovered from the culture medium. Standard techniques of molecular biology are used to prepare the recombinant expression vector, by transfecting the host cells, to select the transformants, to culture the host cells, and to recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or a Protein G. Antibodies can also include modifications, for example, modifications that alter Fe function, for example, to decrease or eliminate interaction with a Fe or Clq receptor, or both. For example, the constant region of human IgGl can be mutated into one or more residues, for example, one or more of residues 234 and 237, for example, according to the numbering in U.S. Patent No. 5,648,260. Other example modifications include those described in U.S. Patent No. 5,648,260. For some antibodies that include an Fe domain, the system for antibody production it can be designed to synthesize antibodies in which the Fe region is glycosylated. For example, the Fe domain of the IgG molecules is glycosylated in asparagine 297 in the CH2 domain. This asparagine is the site for modification with biantennary oligosaccharides. This glycosylation participates in the effector functions produced by the complementary DFcD and Clq receptors (Burton and Woof (1992) Adv. Immunol. 51: 1-84; Jefferis et al. (1998) Immunol Rev. 163: 59-76). The Fe domain can be produced in a mammalian expression system that suitably glycosylates the residue corresponding to asparagine 297. The Fe domain can also include other post-translational eukaryotic modifications. The antibodies can also be produced by a transgenic animal. For example, U.S. Patent No. 5,849,992 describes a method for expressing an antibody in the mammary gland of a transgenic mammal. A transgene is constructed that includes a milk-specific promoter and the nucleic acid sequences encoding the antibody of interest, for example, an antibody described herein, and a signal sequence for secretion. The milk produced by females of these transgenic mammals include, secreted therein, the antibody of interest, for example, an antibody described herein. The antibody can be purified from milk, or for some applications, it is used directly. The antibodies can be modified, for example, with an entity that improves their stabilization and / or retention in circulation, for example, in blood, serum, lymph, bronchoalveolar lavage, or other tissues, for example, in at least 1.5, 2. , 5, 10, or 50 times. In one example, a binding antibody with KIM-1 can be associated with a polymer, for example, a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having average molecular weight weights ranging from about 200 to 35,000 daltons (or about 1,000 to 15,000, and 2,000 to about 12,500) can be used. In another example, a binding antibody with KIM-1 can be conjugated with a water soluble polymer, for example, a polyvinyl hydrophilic polymer, for example polyvinyl alcohol polyvinyl pyrrolidone. A non-limiting list of these polymers includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylene treated polyols, copolymers thereof and block copolymers thereof, provided that the solubility in water from the block copolymers. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and polyoxyethylene and polyoxypropylene block copolymers (Pluronics); polymethacrylates; carbomers; branched or unbranched polysaccharides comprising the saccharide monomers D-mannose, D and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid ( for example polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins, glycogen, or the subunit of polysaccharides of acidic mucopolysaccharides, for example, hyaluronic acid; polymers of alcohols of sugar such as polysorbitol and polymannitol; Heparin or heparin.

Uses and methods of administration In the methods described herein, an agent, such as an antibody that binds to a particular region of KIM-1, is administered to a subject to treat a condition caused by Th2 or a condition caused by Thl. . The treated subject is a mammal, for example, a human being. The "administration" is not limited to any particular formulation, delivery system, or route and may include, for example, intrabronchial, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection) rectal, topical, transdermal, or oral (e.g., in capsules, suspensions, or tablets). The administration can be provided in a single dose or repeatedly, and in any of a variety of pharmaceutical compositions containing physiologically acceptable salt forms, and / or with acceptable pharmaceutical excipients. The physiologically acceptable salt forms and pharmaceutical formulations and excipients are known (see, for example, example, 2004 Physicians' Desk Referent® (PDR) (2003) Thomson Healthcare, 58th. ed; Gennado et al., (2000), 20ava. ed, Lippincott, Williams & Wilkins) Remington: The Science and Practice of Pharmacy. A number of therapeutic agents are useful in the management and treatment of asthma. These include, but are not limited to: bronchodilators, for example, anticholinergic bronchodilators for relaxing the respiratory tract (for example, ipratropium bromide, albuterol bromide / ipratropium); beta agonists to relax the muscles of the respiratory tract (eg, epinephrine, metaproterenol, terbutaline, isoetarinmesilato, isoetarin, isuprel, pirbuterol, albuterol, salmeterol, bitolterol); oral or inhaled corticosteroids to reduce inflammation (eg, hydrocortisone, cortisone, dexamethasone, prednisolone, prednisone, methylprednisolone, flunisolide, triamcinolone, beclomethosone, dexamethasone, fluticasone, budesonide); leukotriene modifiers to prevent the airways from swelling and block airflow and decrease mucus production (eg, zafirlukast, montelukast sodium, zileuton); and theophylline, which helps, among other things, to open the airways and reduce the release of phlegm Anti-asthma agents also include therapeutic antibodies (or functional fragments thereof) including, but not limited to: anti-IgE, anti-IL-9, anti-IL-3, anti-IL-4, anti-IL-5, anti -IL-13, anti-VLA, and anti-migration inhibitory factor (MIF). An antibody described herein may be co-administered with one or more of the above-mentioned agents to treat allergic asthma. Therapeutic agents that are useful in the management and treatment of inflammatory conditions produced by Thl include anti-inflammatory compounds, for example, steroids and NSAIDs. Therapeutically effective dosages achieved in an animal model can be converted for use in another animal, including humans, using known conversion factors (See, for example, Freireich et al (1966) Cancer Chemother, Reports, 50 (4): 219- 244. The following examples provide illustrative modalities The Examples in no way limit the invention Someone with ordinary skill in the art will recognize that many modifications and variations can be made within of the scope of the present invention. These modifications and variations are therefore encompassed by the invention.

EXAMPLES Example 1; Characterization of rat monoclonal antibodies to mouse KIM-1 Using standard PCR and cloning techniques, full-length extracellular domain and murine KIM-1 expression constructs were generated from only the IgV domain and stably transfected into CHO cells . These fusion proteins were purified from supernatants of the CHO cell line by protein A and SEC chromatography. The full-length KIM-1 was fused to a human IgG1-Fc domain that appeared as a doublet, consistent with differential glycosylation. The rats were immunized with the full-length mouse KIM-l-Ig fusion protein consisting of the total extracellular domain. A panel of rat monoclonal antibodies for mKIM-1 was identified by ELISA analysis and FACS selection, and a set of these were additionally characterized by Biacore and by domain-specific ELISA and protein analysis.

Western blotting, which shows that multiple antibodies that bind different epitopes were represented on the panel. In this way, 7 antibodies bound by the full-length protein in Biacore and ELISA analysis, while 4 of these 7 were unable to bind to a protein encoding the IgV domain only (Table 1). Of the 4 antibodies that appeared to require the presence of mucin stem domains to bind in the Biacore format, 3 were further defined by ELISA and Western blot analysis to bind within the mucin domain, while 1 joined in the stem domain (Table 1). Within the mucin domain several antibodies recognized a different region encoded by exon 4 (Table 1). In this way, antibodies that recognize the IgV, mucin, and stem domains were identified. Table 1 shows the results of the epitope mapping of the rat mAb-anti-mKIM-1 mAb. The data in Table 1 were compiled from multiple analyzes (Biacore, ELISA, Western blot, and FACS) using the total ECD of KIM-1-Ig, KIM-1-IgV-Ig, and protein proteolytic fragments. KIM-l-Ig.

TABLE 1 * Predicted Example 2; Induction of KIM-1 expression in hyperactive lung Balb / c mice were primed with OVA / alum twice, then allowed to rest for 3 weeks, at which time the mice received 3 days of aerosol exposure with OVA using a nebulizer. The lung tissue, the drained (bronchial) lymph node and the spleen were collected and examined for the induction of KIM-1 expression by RT-PCR. The KIM-1 message was induced in both bronchial and pulmonary LN tissue for 24 hours after nebulization. In In contrast to KIM-1 mRNA levels, KIM-3 mRNA levels were not modulated after inoculation with the OVA aerosol. KIM-2 mRNA levels were over-regulated in a manner similar to KIM-I.

Example 3; Effect of anti-KIM-1 antibodies on OVA-induced hypersensitivity. Anti-KIM-1 antibodies with different epitope specificities were tested for the ability to influence the development of lung inflammation using the OVA aerosol model, and using both prophylactic and therapeutic dosing regimens. For prophylactic studies, pulmonary inflammation and OVA-induced annulment analyzes were performed as follows: Balb / c mice were administered ip injections of lOOμl 0.5mg / ml OVA (grade V, Sigma) mixed with lOOμl of ImjectAlum (Pierce, Rockford IL USA) on days 1 and 7. Three weeks after the second injection, the mice were exposed for 20 minutes daily for 3 days to a 1% OVA aerosol in PBS using an ultrasonic nebulizer (Devilbiss, Carlsbad CA USA). The dosage with the mAbs was as follows: 200μg were administered IP on days 1, 3, 6, and 9, and then 500μg were administered IP the day the nebulizations started. For therapeutic studies, the mice were immunized with OVA in alum as previously described, although no mAb was administered just before the nebulization series: in this way, 250μg of mAb 4A2 was administered the day before the first nebulization, and 250μg of 4A2 mAb was administered on the morning of the second nebulization. In both prophylactic and therapeutic studies, two days after the final nebulization session, the mice were sacrificed for analysis. The bronchial lavage fluid (BAL) was collected via tracheotomy using 3 washes with PBS containing 0.1% BSA and 0.02mM EDTA. The BAL cells were agglomerated using cytospin and coated platens (Shandon, Pittsburgh, PA USA) then air-dried and stained with Hema3 stain (Fisher Scientific, Pittsburgh PA USA) for the identification of various cell populations. Lung tissue was collected in neutral buffered formalin for routine histology, or frozen instantaneously in trizol for later RNA isolation. The draining (bronchial) lymph nodes and the spleen were collected for the isolation of mononuclear cells, which were placed in culture in RPMI / 10% FBS in varying concentrations of OVA. 72 hours after the supernatants were harvested and the cells were pulsed for 8 hours with thiidine treated with tritium IuCi (Amersham Biosciences, Piscataway, NJ USA) and the plates were counted using the Microbetajet system (Wallac, Gaithersburg, MD USA). The supernatants were analyzed using CBA Thl / Th2 and the equipment for Inflammation (BD Biosciences) and ELISA IL-13 analysis (R & D Systems, Minneapolis MN USA).

Results: The mice were dosed with antibodies during the priming and inoculation phases with OVA. After inoculation, bronchial lavage fluid (BAL), bronchial lymph node, spleen, and lung tissue were collected. The percentages of eosinophils, neutrophils, and lymphocytes present in BAL were calculated. MAb 1H8 induced approximate counts of eosinophils in the BAL of treated mice, such that thepercentage of eosinophils present was more than double with respect to the control. Modest increases were also observed in the percentage of neutrophils and lymphocytes, which constitute a small fraction of BAL cellularity. Consistent with this result, LN bronchial cells isolated from mice treated with 1H8 and inoculated with OVA ex vi vo proliferated further, and expressed higher levels of cytokines associated with Th2 than control cultures. In particular, very high levels of IL-5 and IL-13 were produced, compared to controls, although levels of IL-4, IL-6, and IL-10 were also raised. Of interest, the levels of IFN-gamma were also increased, although in general, the levels of this cytokine were low. The preferential induction of the Th2 cytokines could be effective in the adjustments of the pathology dependent on the Thl cytokine, such as MS, RA, Crohn's. In contrast to 1H8, mAb 3A2 reduced the percentage of eosinophils in the BAL, and reduced the production of the cytokines associated with Th2 in the analysis of cancellation of bronchial lymph nodes (Fig. 2). In this way, antibodies 1H8 and 3A2 have opposite effects in this analysis. Various other antibodies, including 1H9, which recognizes an epitope within the IgV domain, had no effect on lung inflammation or cytokine production in this model. The analysis of the drainage (bronchial) response of lymph node cells for ex vivo stimulation with the OVA antigen with 3A2 showed a reduction in cell proliferation for stimulation with antigens (Fig. 3). In addition, the analysis of the supernatants from these cultures showed a marked reduction in the expression of the Th2 cytokines including IL-4, IL-5, and IL-10 (Fig. 4). In this analysis BL-13 levels were also reduced. Treatment with the mAb 4A2 of the anti-Ig domain resulted in a marked reduction in the influx of eosinophils and lymphocytes in the BAL after sensitization with OVA treatment and with nebulization (Fig. 6). The average decrease in the percentage of eosinophils in the BAL was 84% compared to the control (p <0.0001, medium equivalence test) and the average decrease in the percentage of lymphocytes was 90% compared to the control ( p <0.001, medium equivalence test). When the bronchial cells of the lymph node were stimulated again with OVA ex vi vo there was a decrease dramatic in the production of Th2 cytokines including IL-4, IL-5, IL-10, and IL-13 (Fig. 7). Therefore, treatment with mAb 4A2 reduced lung inflammation and cytokine production associated with asthma responses. To further characterize the clinical efficacy of mAb 4A2, a therapeutic dosing experiment was performed. In this model, the mice were immunized to develop sensitivity to the OVA antigen, without administering any treatment with the mAb. The mice were then allowed to rest for 3 weeks, again without any treatment, and then dosed with mAb 4A2 the day before the first of 3 sessions of nebulization with 1% OVA. The dosage with the mAb was repeated before the second session. This treatment protocol resulted in the reduction of lung inflammation as measured by the influx of eosinophils in BAL (Fig. 8). The percentage of eosinophils was reduced by an average of 70% (p <0.001, test of the mean equivalence). Therefore mAb 4A2 was effective in a prophylactic and therapeutic dosing regimen in the model of OVA-induced lung inflammation. This suggests that the epitope recognized by 4A2 is a target Therapeutically relevant for the treatment of disorders produced by Th2. It was also shown that other anti-KIM-1 mAbs have therapeutic activity in the OVA-induced lung inflammation model, including, for example, mAb 2A7 and mAb 2B3. The 2A7 mAb was shown to compete with 4A2 for binding with immobilized KIM-1 in a Biocore® analysis, suggesting that they have shared or overlapped epitopes.

Example 4; Effect of KIM-1 antibodies on the response of CD4 T lymphocytes to the antigen The activity of anti-KIM-1 mAbs was evaluated using the antigen KLH. The mice were treated with the anti-KIM-1 mAb, control mAb, or PBS, then immunized with KLH and 6 days after draining with LN were subjected to extirpation. The CD4 + LN T lymphocytes were isolated and ex vivo stimulated with purified OVA in the presence of irradiated whole splenocytes isolated from untreated mice. 48 hours after ex vivo stimulation, cell proliferation and cytokine production were tested. In this analysis several of the anti-KIM-1 mAbs had a marked effect. MAb 1H8 dramatically increased the proliferation of T lymphocytes in response to the exogenous inoculation of KLH. In contrast, mAb 3A2 reduced the proliferation of T lymphocytes in the analysis. The cytokines produced in the cultures from the cells treated with mAb 1H8 were measured. It was found that the treated cultures contained more cytokines IFN-gamma and TH2 -associated than the controls. In contrast, the levels of TNF and IL-2 were similar to the controls. These data indicate that 1H8 can reduce the pathogenic Thl response. These data also indicate that 1H8 and other antibodies that bind to the KIM-1 region as defined herein for 1H8 may act as adjuvants by increasing the immune response. The invention also covers methods for increasing the immune response, for example, to increase the efficacy of a vaccine. This adjuvant can also be used in vaccination, immunodeficiency, and anti-tumor immunity. 1H8 binds to the KIM-1 ECD of the Balb / C sequence but not to the Dba / 2 sequence in Western blots. This indicates that the antibody binds to the mouse allelic variant alternatively assembled containing the sequence EPTTFCPHETTAEVTGIPSHTPT (SEQ ID NO: 2). This sequence corresponds to the sequence VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT of human KIM-1 (amino acids 200-235 of SEQ ID NO: 1).

Example 5; Characterization of mAb 3A2 Because 3A2 mAb had a therapeutic effect in the OVA model, its binding to KIM-1 was characterized in greater detail. Various purified proteins were used in an ELISA analysis to determine the epitope of mAb 3A2 (Table 2). The total binding curves were generated for the interaction of 3A2 with the immobilized proteins (Fig. 5). MAbs 3A2 were equivalently linked to the following mouse proteins: extracellular domain of murine KIM-1 (KIM-1-ECD-1-216), mKIM-1 -137-216 and mKIM-1-196-216-Fe. In contrast, mAb 3A2 could not bind to the mKIM-1-IgV domain alone, which is lacking the total domain of mucin and stem. These data show that the epitope for 3A2 resides within 21 amino acid residues 196 to 216 of mKIM-1, which maps to a portion of the stem region of KIM-1. East epitope is equal to residues 247-272 of human KIM-1 as shown in Fig. 1.

TABLE 2 Because the stem region that includes amino acids 196 to 216 of mouse KIM-1 (corresponding to amino acids 247-272 of human KIM-1) contains the N-glycosylation sites, it was interesting to determine if it was required for the 3A2 binding a sugar entity attached to an N-glycosylation site. An analysis of Western blot of KIM-1-196-216 - Glucosylated and deglycosylated fiber (Figure 2). This analysis showed that deglycosylation did not affect the ability of 3A2 to bind to KIM-1-196-216-Fc. Therefore, a sugar entity for 3A2 was not required to recognize its epitope. To determine whether 3A2 inhibits KIM-1 spillage from the cell surface, E293 cells transfected with KIM-1 were treated with 5μg / mL of 3A2, 25μg / mL of 3A2 or no 3A2 (control). The supernatants from both sets of cells treated with 3A2 showed no difference in staining with KIM-1 from the control when run on the Western blot in test solution with a biotinylated 1H8 antibody. This suggests that 3A2 does not prevent the spill of KIM-1.

Example 6. Characterization of mAb 4A2. Using ELISA and Biacore analysis, it was determined that mAb 4A2 recognized the Ig domain of murine KIM-1 (Fig. 9, Table 1). To further characterize the epitope recognized by mAb 4A2, an IgV-human Fe IgGl fusion of recombinant murine KIM-1, alone and in complex with 4A2, was digested with TPCK trypsin. An 8kDa band of KIM-1 was generated alone and was not generated when it joined 4A2. This indicates that the binding of 4A2 to KIM-1 blocks the access of trypsin to the cleavage site required to generate this band. The 8Kda band was reduced and subjected to further cleavage with chymotrypsin and mass spectrometry analysis that produced the sequences corresponding to the peptide coverage of amino acids 2 to 103 of murine KIM-1. This is an amino acid fragment whose C term corresponds to a cleavage site with chymotrypsin that is adjacent to a trypsin cleavage site in the region identified to have homology to the sialic acid binding region, which is for the sequence CRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: l). In this way, the 4A2 antibody protects a TPCK trypsin site at least partially within, or they overlap with the amino acids 81-107 of SEQ ID NO: 1. The same tryptic digestion experiment of TPCK was performed with 2A7 and a band of approximately the same size was obtained. The band was not obtained when the same experiment was performed with 1H9 (a mAb not effective in the asthma model that also binds to the Ig domain), indicating that the epitope is effectively tracked in asthma. The specification will be understood more fully in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of the embodiments of the invention and the scope of the invention should not be construed as limiting. The skilled person will readily recognize that many other embodiments are encompassed by the invention. All publications, patents, and biological sequences cited in this exhibit are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification will replace any of these materials. The citation of any references herein is not an admission that these references are the prior art of the present invention. Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, etc., used in the specification, including the claims, should be understood to be modified in all cases by the term "approximately". Accordingly, unless otherwise indicated, the numerical parameters are approximations and may vary depending on the desired properties intended to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that these equivalents be encompassed by the following claims.

Claims (46)

1. CLAIMS 1. A method for treating a disorder caused by Th2 in a mammal, the method comprising administering to the mammal an antibody, or a binding fragment with antigens thereof, that binds to the stem region of KIM-I.
2. 2. The method according to claim 1, wherein the mammal is a human being.
3. 3. The method according to claim 1, wherein the disorder is atopy.
4. 4. The method according to claim 1, wherein the disorder is asthma.
5. 5. The method according to claim 1, 2, 3 or 4, wherein the antibody binds to an epitope at least partially contained in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1).
6. 6. The method according to claim 1, 2, 3 or 4, wherein the antibody is a monospecific humanized or fully human antibody.
7. 7. The method according to claim 1, 2, 3 or 4, wherein the antibody is a humanized or fully human monospecific antibody and wherein the antibody binds to an epitope at least partially contained in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1).
8. 8. The method according to claim 1, 2, 3, or 4, wherein a full-length antibody is administered.
9. 9. The method according to claim 1, 2, 3, or 4, wherein a full-length antibody is administered and wherein the antibody binds to an epitope at least partially contained in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO. NO: 1).
10. 10. The method according to claim 1, 2, 3 or 4, wherein a binding fragment is administered with antigens of an antibody.
11. 11. The method according to claim 1, 2, 3 or 4, wherein a binding fragment is administered with antigens of an antibody and wherein the antibody binds to an epitope at least partially contained in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1).
12. 12. The method according to claim 10, wherein the antigen binding fragment is selected from the group consisting of: single chain antibodies, Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, and dAb fragments.
13. 13. The method according to claim 11, wherein the antigen binding fragment is selected from the group consisting of: single chain antibodies, Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, and dAb fragments.
14. 14. The method according to claim 1, 2, 3 or 4, wherein the antibody or antigen-binding fragment is administered in combination with a second therapeutic agent for the disorder.
15. 15. The method according to claim 1, 2, 3 or 4, wherein the antibody or antigen-binding fragment is administered in combination with a second therapeutic agent for the disorder and wherein the antibody binds to an epitope contained at least partially in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1).
16. 16. The method according to claim 1, 2, 3 or 4, wherein the antibody or antigen-binding fragment is administered at a dosage between 0.05 and 20 mg / kg.
17. 17. The method according to claim 1, 2, 3 or 4, wherein the antibody or antigen binding fragment is administered at a dosage between 0.05 and 20 mg / kg and wherein the antibody binds to an epitope at least partially contained in the peptide DGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTK (amino acids 236-269 of SEQ ID NO: 1).
18. 18. An isolated antibody, or antigen-binding fragment thereof, that specifically binds the stem region of KIM-1, in where the antibody does not inhibit KIM-1 efflux from E293 cells in culture.
19. 19. The method according to claim 1, 2, 3 or 4, wherein the antibody binds to a peptide having a sequence selected from the group consisting of: (a) amino acids 262-270 of SEQ ID NO: 1, (b) ) amino acids 260-269 of SEQ ID NO: 1, (c) amino acids 257-270 of SEQ ID NO: 1, (d) amino acids 252-270 of SEQ ID NO: 1, (e) amino acids 236-250 of SEQ ID NO: 1, and (f) amino acids 236-258 of SEQ ID NO: 1.
20. 20. The method according to claim 19, wherein the antibody is a monospecific antibody humanized or fully human.
21. 21. The method according to claim 19, wherein a full-length antibody is administered.
22. 22. The method according to claim 19, wherein a binding fragment is administered with antigens of the antibody.
23. 23. The method according to claim 19, wherein the antibody or antigen-binding fragment is administered in combination with a second therapeutic agent for the disorder.
24. 24. The method according to claim 19, wherein the antibody or antigen binding fragment is administered in a dosage between 0.05 and 20 mg / kg.
25. 25. A method for treating a disorder caused by Thl or for reducing a pathogenic response to Thl in a mammal, the method comprises administering to the mammal an antibody, or antigen-binding fragment thereof, that binds to the sequence VATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTT of KIM-1 human (amino acids 200-235 of SEQ ID NO: 1) or an epitope that overlaps the sequence.
26. 26. The method according to claim 25, wherein the mammal is a human being.
27. 27. The method according to claim 25, wherein the antibody is a monospecific antibody humanized or fully human.
28. 28. The method according to claim 25, wherein a full-length antibody is administered.
29. 29. The method according to claim 25, wherein a binding fragment is administered with antigens of the antibody.
30. 30. The method according to claim 25, wherein the antibody or antigen-binding fragment is administered in combination with a second therapeutic agent for the disorder.
31. 31. The method according to claim 25, wherein the antibody or antigen binding fragment is administered in a dosage between 0.05 and 20 mg / kg.
32. 32. A method of treating a Th2-caused disorder in a mammal, the method comprises administering to the mammal an antibody, or antigen binding fragment thereof, that binds to the sialic acid binding motif of KIM-1.
33. 33. The method according to claim 32, wherein the antibody binds at least partially to a epitope contained within or superimposed on a peptide having the sequence GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1).
34. 34. The method according to claim 32, wherein the antibody binds to a linear epitope of contiguous amino acid residues of GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) or RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO: 1) .
35. 35. The method according to claim 32, wherein the antibody binds to a structural epitope to which one or both of the sequences GVYCCRVEHRGWFNDMKITVSLEIVPP (amino acids 81-107 of SEQ ID NO: 1) and RGSCSLFTCQNGIV (amino acids 29-42 of SEQ ID NO. : 1) .
36. 36. The method according to claim 32, wherein the antibody protects amino acids 81-107 of SEQ ID NO: 1.
37. 37. The method according to claim 32, wherein the antibody interferes with one or more of the residues R86, W92, and F93 of SEQ ID NO: 1.
38. 38. The method according to claim 32, 33, 34, 35, 36 or 37, wherein the mammal is a human being.
39. 39. The method according to claim 32, 33, 34, 35, 36 or 37, wherein the disorder is atopy.
40. 40. The method according to claim 1, wherein the disorder is asthma.
41. 41. The method according to claim 32, 33, 34, 35, 36 or 37, wherein the antibody is a monospecific humanized or fully human antibody.
42. 42. The method according to claim 32, 33, 34, 35, 36 or 37, where a full-length antibody is administered.
43. 43. The method according to claim 32, 33, 34, 35, 36 or 37, wherein a binding fragment is administered with antigens of the antibody.
44. 44. The method according to claim 43, wherein the antigen binding fragment is selected from the group consisting of: single chain antibodies, Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, and dAb fragments.
45. 45. The method according to claim 32, 33, 34, 35, 36 or 37, wherein the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent for the disorder.
46. 46. The method according to claim 32, 33, 34, 35, 36 or 37, wherein the antibody or antigen-binding fragment thereof is administered at a dosage between 0.05 and 20 mg / kg.