MXPA00008040A - Treating and diagnosing macrophage-mediated diseases using fc receptor ligands - Google Patents

Abstract

The invention provides methods and compositions for selectively targeting macrophages in a localized area. The compositions of the invention include an Fc receptor binding agent, and a toxic or a detectable agent. Methods for depleting or inhibiting the activity of macrophages using the compositions of the invention are disclosed. The compositions of the invention can be used therapeutically and diagnostically.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF MACROFAGOS MEDIATED DISEASES BACKGROUND OF THE INVENTION In normal human skin it is possible to distinguish two compartmental layers. The upper layer, the epidermis, consists of keratinocytes, Langerhans cells and T cells. The lower layer, the dermis, consists of fibroblasts, endothelial cells, dendritic cells, T cells, mast cells and macrophages. The skin serves as an important barrier between the internal environment and the environment. It mainly avoids contact with potentially dangerous antigens. In case of antigen / pathogen penetration, an inflammatory response is induced in vivo to eliminate the antigen. This response leads to a dermal infiltrate, the composition of which depends on the type of response induced, but consists mainly of T cells, polymorphonuclear cells and monocytes (Williams, IR and Kupper, TS (1996), Life Sci. 58: 1485-1507 Stingl, G. (1993) Recent Resul ts Cancer Res. 128: 45-57). In addition, non-specific allergen stimuli such as tissue injury and ultraviolet light can also activate an inflammatory response. In general, the mechanisms underlying the non-specific response of the allergen are also they are used during the effector phase of the allergen-specific response. The macrophages with cells that come from the bone marrow with great heterogeneity and versatility. These cells can produce a wide range of mediators and produce a multitude of biological functions (Ganz, T. (1993) New hori z. 1: 23-27). Its phenotype and function is determined to a large extent by the local environment, while mediators from macrophages can, on top of this, influence its microenvironment. This micro-environment allows subsets of regionally different macrophages and subsets of locally different macrophages to be present (Gordon, S. (1995) Bioessays 17: 977-986). These cells are powerful effector cells that produce reactive oxygen products and proteolytic enzymes, which can directly damage tissue (Laskm, D. L. and Pendino, K. J. (1995) Annu. Rev Pharmacol. Toxicol 35: 655-677). Under normal conditions, macrophages regulate the proliferation of extracellular matrix-forming cells such as fibroblasts in the skin (Gonzales-Ramos, A. et al., (1996) J. Invest. Dermatol. 106: 305-311). In addition, macrophages can exert important immunoregulatory functions and thus play an important role in the control and direction of immune responses (Gordon, S. (1995) Bioessays 17: 977-986; Thepen, T. et al., ( 1994).

Ann N. Y. Acad Sci 725: 200-206). These cells can serve as antigen-presenting cells, but also directly inhibit antigen presentation by dendritic cells (Holt, P. G. et al., (1993) Exp. Med 177: 397-407). The proliferation, phenotype and thus the function of the T cells, and by this means the type of induced immune response, can be influenced by the macrophages.

It has been shown that skin macrophages play a role in the regulation of cell growth of different non-hematopoietic cells (such as fibroblasts and keratinocytes), as well as in the functioning of T cells and dendritic cells. Under "steady state" conditions, the number of macrophages in the skin is relatively low. However, under different pathological conditions (for example, in active lesions), the number of macrophages grows significantly. Infiltrating monocyte and tissue macrophages have been associated with the modified function of fibroblasts and keratinocytes in inflammatory lesions, as well as aberrant functioning of T cells and / or dendritic cells. It has been shown that exposure to ultraviolet light induces a population of macrophages in the skin that, unlike Largerhans cells, are capable of activating self-reactive T cells. The deregulated function of Macrophage has been directly correlated with abnormal cutaneous immune response in different diseases, including cutaneous T-cell lymphoma (mycosis fungoides), psoriasis, atypical dermatitis and cutaneous lupus erythematosus (Koper, KD et al., (1993) J. Invest. Dermatol 101: 155-163; González Ramos A. et al., (1996) J. Invest. Dermatol 106: 305-3ll). These cells can also activate resident and inflammatory macrophages, giving rise to a "vicious circle" that maintains skin inflammation. In addition to the regulation of cellular function, macrophages are potent products of toxic compounds such as oxygen radicals and proteolytic enzymes. These toxic compounds cause direct damage to the tissue.

SUMMARY OF THE INVENTION The present invention provides methods and compositions for selectively targeting cytotoxic compounds through Fc receptors to phagocytic cells derived from monocytes (ie, macrophages). The invention can thus be used to selectively reduce the number or activity of a population of macrophages within a localized area, such as the skin, joints or lungs. In one embodiment, the invention provides a compound that binds to the macrophage, which contains at least one first portion that binds to an Fc receptor present in a macrophage, and at least a second portion that kills or inhibits the function of the macrophage. The portion that binds to the Fc receptor can include any molecule capable of binding to the Fc receptor, such as an antibody, a peptide (e.g., mimetic peptide) or a chemical compound. In one embodiment, the portion that binds to the Fc receptor is an antibody or antibody fragment (e.g., a Fab, Fab ', F (ab'> 2, F or monocatheter Fv.) In a preferred embodiment, the antibody or Fc antireceptor antibody fragment is "humanized" (for example, it has at least one complementarity determining region (CDR) or a portion thereof from a non-human antibody (e.g., from Murino) with the (s) remnant portion (s) of human origin.) In another preferred embodiment, the antibody, or anti-Fc receptor antibody fragment, is a human monoclonal antibody (e.g., an antibody produced in a genetically engineered mouse to express an antibody completely human) Also included among these modalities are the compounds or example, peptides or chemical species) that "mimic" the binding of these anti-Fc receptor antibodies (Jenks et al., J. Nati. Cancer Inst (1992) 84 (2): 79; Saragovi et al., Science (1991) 253: 792; Hinds et al., J. Med. Chem. (1991) 34: 1777-1789; Fassina Immunomethods (1994) 5: 121-129). In another modality, the portion that binds to the Fc receptor of the compound that binds to the macrophage is a cyanine composition, such as the fluorescent dye Cy5.18.Osu (mentioned herein as "Cy5", which binds with great affinity and specificity to FcγRI receptor present in the macrophage cells The cyanine compositions can include at least two portions: a cyaninsuccinimidyl ester and a phycobilisome protein, eg PE In the Fc receptor recognized by the compounds that bind to the macrophage of the invention to be an IgG receptor, for example, an Fc-gamma receptor (Fc? R), such as Fc? RI (CD64), Fc? RII (CD32) and Fc? RIII (CD16), or an IgA receptor, for example , an Fc R (for example, Fc? RI, CD89) The Fc receptor is preferably located on the surface of a macrophage, for example, a skin macrophage, so that it is capable of being recognized and bound by the compound In a preferred embodiment, the portion that binds to the anti-Fc receptor of the compound that is ne to the macrophage binds to an Fc receptor at a site other than that which was combined by endogenous immunoglobulins (eg, IgG or IgA). Therefore, the combination of the compounds that bind to the macrophage to the Fc receptor is not blocked by the physiological concentrations of the immunoglobulins. A preferred Fc receptor in a macrophage for the choice is the Fc receptor? high affinity, Fc? RI. A) Yes thus, in one embodiment, the portion that binds to the anti-Fc receptor of the compounds that bind to the macrophage of the invention consists of an anti-FcγRI antibody or a fragment thereof. Exemplary anti-Fc? RI antibodies include mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. In preferred embodiments, a humanized form of these antibodies that bind to the anti-Fc? RI receptor is used, as to be the humanized monoclonal antibody 22 (H22), or a fragment thereof. The portion of the compound that binds to the macrophage that kills or modulates (e.g., reduces) the activity of a macrophage (the anti-macrophage agent) can be selected from cytotoxins or convenient drugs. For example, the anti-macrophage agent can be Gelonin, Saporm, Onconase, Exotoxin A, Ricin A, dichloromethylene diphosphonate (CL2MDP) or derivatives thereof. In one embodiment, the anti-macrophage agent binds directly to the anti-Fc receptor combination portion. For example, the anti-macrophage agent may be encapsulated with a liposome which binds to the anti-Fc receptor combination portion. The compounds that bind to the macrophage of the invention can be used in a variety of therapeutic and diagnostic methods. In one embodiment, these compounds are used to diagnose a disease characterized by abnormal numbers or functions of the macrophages. The method includes contacting or administering to a test area or cultured sample, the compound that binds to the macrophage under conditions that allow the combination of the compound to the macrophages present in the sample. The binding of the compound can then be detected as an indication of the presence (for example, the number) and / or the function of the macrophages in the sample. For example, a statistically significant high concentration of the specifically detected Fc receptor protein, indicating an increase in the number of macrophages, may be indicative of a disease. The test area or sample may be from, for example, the skin (e.g., human skin) or other tissue containing macrophage cells. In another embodiment, the compounds that bind to the macrophages are used to treat a disease that includes the proliferation and / or abnormal functioning of the macrophages. With the contact of the compounds that bind to the macrophages with an area that needs treatment, the compounds bind to the macrophages through their Fc receptors and annihilate or reduce the activity of these cells. Therefore, a wide variety of diseases that include macrophages (for example, proliferation and / or abnormal functioning of macrophages) can be treated, avoided or diagnosed using the compounds of the invention. These diseases may be intrinsic origin (eg, autoimmune disease I), or of extrinsic origin (eg, hypersensitivity to contact), Polymorphic Light Eruption 5 (PLE), and irritant reactions). Skin disease can also be a manifestation of a more systemic disease such as atopic dermatitis (AD) in the case of atopy, and lupus e? Tematoso systemic. A non-limiting list of diseases that can be treated with the compositions and methods of the present invention include autoimmune diseases, respiratory diseases, infectious diseases, dermatological diseases and inflammatory conditions. Specific examples of these diseases include, but are not limited to: psoriasis, atopic dermatitis, multiple sclerosis, scleroderma, cutaneous lupus e? Tematoso, rheumatoid arthritis, infections of Human Immunodeficiency Virus (HIV), Chronic Polymorphic Dermatosis Mild (CPLD), Chronic Obstructive Lung Diseases (DOPD) for example, allergic asthma and sarco-dose, Wegener's granulomatosis, and inflammatory conditions such as skin lesions (eg, open wounds or burns). In addition, the methods and compositions of the invention can be used to diagnose such diseases, or for the purposes of research (for example, to study the pathological function of macrophages in such diseases). When used in vivo for therapeutic purposes, the compounds bound to the macrophages of the invention can be administered locally (e.g., topically, intradermally, subcutaneously or by inhalation as in an aerosol) in a selected area in a effective amount to deplete or reduce the activity of the macrophages within the administration area. In certain embodiments, the compound that binds to the macrophage may include a photosensitizing agent that is inactive when administered (eg, systemically, topically, intramuscularly) but is activated by exposure to light (e.g., visible light). or UV light). In the same way, the compounds that bind to the macrophages can include an agent that binds to Fc linked to a therapeutic or diagnostic reagent through a photo-dissociable bond, which upon exposure to light releases the reagent. These compounds allow controlled killing or inactivation of macrophages only within selected tissues exposed to light.

The present invention furthermore offers compositions, for example, pharmaceutical compositions, containing compounds that bind to macrophages together with an acceptable carrier or diluent, for use in the methods described above. -_______ i___.

Other features and advantages of the invention will be apparent from the following figures, the detailed description, the examples and the clauses.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar graph representing the percentage of [H] -thymidine incorporation of cultured U937 or IIA1.6 cells grown in the presence or absence of different concentrations of CD64-immunotoxin (H22- ric? n A, H22-R or 197-Ricin A, 197-R) compared to that of a medium control (± SEM). U937 cells were cultured with (black bars) or without (gray bars) IFN? in the presence of indicated concentrations of H22-R (panel A) or 197-R (panel B). In the lower panels C and D, the IIA.16 cells transfected with hFc? RI (black bars) or non-transfected cells (gray bars) were incubated with different concentrations of H22-R (panel C) or 197-R (panel D) . Figure 2 is a sweep of the propidium iodide fluorescence of U937 cells as these cells underwent apoptosis after incubation with different concentrations of H22-R. The nuclear fragmentation was analyzed with propidium iodide staining and the subdiploid nuclei are indicated by bars. The numbers above the bars specify the percentage of the subdiploid nuclei, therefore apoptotic. With = control.

Figures 3A and B are graphs showing the effect of a single intradermal injection of an immunotoxin on inflammatory cells in skin with respect to time. The data points represent the average number of cells 2 per mm (+ SEM) and the data points represent the average of > 3 experiments The kinetics of the cells expressing hFcγRI (frames filled with Figure 3A), macrophages (black boxes, Figure 3A), T cells (filled boxes, Figure 3B), and dendritic cells (black boxes, Figure 3B) are represented. . Figures 4A-4B are graphs showing a decrease in local skin temperature with the intradermal injection of an immunotoxin. Figure 4A depicts local skin temperature readings (± SEM) of transgenic hFc? RI mice treated with SLS after a single injection with IT (•) (n = 6) or vehicle control (0) (n = 6). Figure 4B shows the temperature course (+ SEM) of transgenic hFc? RI mice treated with SLS, injected with IT (0) (n = 6), or vehicle control (•) (n = 6). The local temperature of the skin was monitored daily, with an increase, the animals were injected again in the same place (days marked with *).

DETAILED DESCRIPTION OF THE INVENTION Abnormal function of macrophages, including aberrant proliferation and / or activity, have been implicated in different disorders, such as dermatological diseases, autoimmune diseases, infectious diseases and inflammatory conditions. To date, methods of localized ablation of macrophages using cytotoxic agents, for example, immunotoxins, have had limited efficacy. The present invention provides the methods and compositions for the diagnosis, treatment and prevention of these disorders by depletion and / or selective inhibition of the activity of the macrophages within a localized area. The cells are depleted (eg, annihilated) and / or inhibit (e.g., activity is reduced) by targeting a toxic agent to these through their Fc receptors. For example, the studies described herein demonstrate the use of a compound that binds macrophages consisting of an anti-receptor binding moiety, Fc, eg, a humanized antibody against a human FcγRI receptor, conjugated with a toxin, e.g., Ricin A, to selectively remove macrophages in vivo in transgenic mice expressing human FcγRI. As used herein, the terms "macrophage" and "phagocytic cells obtained from monocytes" should be used interchangeably.

Accordingly, in one embodiment, the invention provides a compound that binds to macrophages and that consists of an agent that binds to an Fc receptor present in a macrophage and an agent that annihilates or inhibits the activity of the macrophages that are bound. Suitable components for binding to Fc receptors, e.g., proteins (e.g., anti-FcR antibodies and peptides or mimetic chemicals thereof, or FcR receptor ligands) and chemical moieties (e.g., synthetic FcR dyes and ligands) . Such agents that bind to the Fc receptor may be monospecific, bispecific or multispecific in that they contain one, two or more than two combination regions, respectively. For example, the agent can bind to two or more different regions of an Fc receptor, or to an Fc receptor and a different component of the same or another cell. In all cases, the agent contains at least one portion that binds to an Fc receptor. In one embodiment, the agent that binds to the Fc receptor is an antibody, or an antibody fragment, which includes, for example, a Fab, Fab ', F (ab') 2, Fv or single chain Fv fragment. The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof such as Fv or a single chain construct as described in Ladner et al., Patent No. 4,946,778, published on August 7, 1990, the content of which is expressly incorporated by reference. In another embodiment, the agent that binds to the Fc receptor is an antibody mimetic (e.g., a peptide or a chemical compound) (Jenks et al., J. Na ti. Cancer Inst. (1992) 84 (2) : 79; Saragovi et al., Science (1991) 253: 792; Hinds et al., J. Med. Chem. (1991) 34: 1777-1789; Fassina Immunomethods (1994) 5: 121-129). In another embodiment, the component that binds to Fc is a bispecific or multispecific molecule. The term "bispecific molecule" is intended to include any compound, for example, a chemical moiety or a protein, peptide or protein or complex peptide, which has two different binding specificities that bind to or interact with: (a) an Fc receptor on the surface of a macrophage, and (b) a second antigen chosen differently. The term "multispecific molecule" or "heterospecific molecule" is intended to include any compound, for example, a chemical moiety, a protein, peptide, or complex protein or peptide, which has more than two distinct binding specificities and which binds to , or interact with (a) an Fc receptor on the surface of a macrophage, (b) two more different antigens chosen. Accordingly, the agents that bind to the Fc receptor that jai'- --- can be used in the compounds that are combined with macrophages of the invention include bispecific, trispecific, tetraspecific, and other multispecific molecules that are directed to the Fc receptors in macrophages. For example, the agent may be a heterobody that contains two or more antibodies, antibody binding fragments (eg, Fab), or derivatives thereof, linked together having different specificities. These different specificities may include two or more different binding specificities in an Fc receptor. Otherwise, they can include a binding specificity on an Fc receptor, and at least one other different binding specificity in the same cell (i.e., a macrophage) or a different chosen cell (e.g., another immune or a pathogen). In such embodiments where the agent that binds Fc is a bispecific or multispecific molecule, the agent can function to physically assemble a cytotoxic effector cell to a chosen macrophage, so that it is obtained directed and more efficient elimination of the macrophage. As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, contrary to the cognitive and activation phases of an immune response. The exemplary immune cells include a cell of an origin myeloid or lymphoid, for example, lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTL)), killer cells, natural killer cells, eosinophils, neutrophils, polymorphonuclear cells, granulosites, mast cells and basophils. Like macrophages, effector cells express specific Fc receptors and perform specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cellular toxicity (ADCC), for example, a neutrophil capable of inducing ADCC. For example, neutrophils, eosinophils and lymphocytes that express Fc? R are involved in the specific death of the chosen cells and present antigens for other components of the immune system, or bind to cells that present antigens. In other embodiments, an effector cell can phagocytose a chosen antigen or cell (e.g., a macrophage), or a microorganism, or it can lyse a chosen cell, e.g., a macrophage. The expression of the particular Fc receptor in an effector cell can be regulated by humoral factors such as cytokines. For example, the expression of FcγRI has been found to be activated by interferon gamma (IFN-γ). This improved expression increases the cytotoxic activity of the FcγRI carrier cells against targets, for example, macrophages.

In other embodiments of the invention, the agent that binds to the Fc receptor is a monoclonal antibody or fragment thereof. The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein, refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition shows a single binding and affinity specificity for a specific epitope. The monoclonal antibody can be murine or human monoclonal antibody (for example, an antibody produced in a mouse genetically engineered to express fully human antibodies). In yet other embodiments of the invention, the agent that binds to the Fc receptor is a chimeric antibody or fragment thereof or a humanized antibody or fragments thereof. A "chimeric antibody" is proposed to include an antibody in which the variable regions are from one species of animal and the constant regions are from another species of animal. For example, a chimeric antibody can be an antibody having variable regions that come from a mouse monoclonal antibody and constant regions that are human. In a preferred embodiment of the invention, the compound that binds to macrophages consists of a humanized antibody or binding fragment thereof. The term "humanized antibody" is proposed to include antibodies in which the hypervariable regions, also called the complementarity determining regions (CDR) are from one animal species and the structural regions and constant regions of the antibody are from animal species of different species. In a humanized antibody of the invention, the CDRs are from a mouse monoclonal antibody and the other regions of the antibody are human. In preferred embodiments, a human antibody is obtained from known proteins NEWM and KOL for the heavy chain variable regions (VH) and REI for the Ig kappa chain, the variable regions (VK). The term "antibody," as used herein, is proposed to include chimeric and humanized antibodies, antibody binding fragments or modified versions thereof. The term "fragment" or "binding fragment" of an antibody or protein capable of binding to an antigen is proposed to include a fragment of the antibody or protein that is sufficient to bind the antigen. The combination of a binding fragment of an antibody to an antigen can be within the same affinity or a different affinity, eg, lower or higher affinity, as the binding of the total antibody to the antigen. Examples of the binding fragments comprised within the term antibody include: a Fab fragment consisting of the domains VL, VH, CL and C?; and the Fd fragment consists of the VH and CHI domains; a Fv fragment consists of the L and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341: 544-546) consists of a VH domain; an isolated complementarity determining region (CDR); and a F (ab ') 2 fragment, a divalent fragment consists of two Fab' fragments linked by a disulfide bridge in the hinge region. A binding fragment, for example, a binding fragment of an antibody can be an active or functional binding fragment. Accordingly, an active or functional binding fragment is proposed to include binding fragments that are capable of activating at least one activity or function activated by the full-length molecule. For example, an active binding fragment of monoclonal antibody M22 or H22 is a fragment of the antibody that is capable of binding to FcγR and activating the activity of the effector cells mediated by the receptor, for example, the production of the superoxide anion. These antibody fragments are obtained using the traditional techniques known to those of ordinary skill in the art, and the fragments are detected for utility in the same way as is done for intact antibodies.

The terms "an agent that binds to" or "binding specificity" are used interchangeably herein with the terms "antigen binding site", "binding region" to the antigen "and" determinant of binding of an antibody. "These terms are proposed to include the region of a molecule, an antibody, that is involved in binding to an antigen.The antigen binding site of an antibody consists of, but it is not limited to the amino acids of the antibody that make contact with the antigen.The antigen-binding region can be the variable region of an antibody.The antigen-binding region of an antibody can also be the hypervariable regions of an antibody. The antigen binding region of an antibody can also be the amino acid residues in the hypervariable region of an antibody that makes contact with the antigen and / or which provides adequate tertiary structure of the antigen-binding region. disposition to determine the amino acid residues of a variable region or hypervariable region of an antibody that makes contact with the antigen and / or that they are important because they have a properly folded antigen binding region. For example, it is possible to perform mutagenesis analyzes, in particular, it is possible to substitute one or more amino acids for other amino acids in the recombinantly produced antibody and perform the binding studies in vi tro to determine the degree to which the binding affinity of the Antibody modified for the antigen has changed compared to the unmodified antibody. If the union has decreased due to the substitution of one amino acid for another, it is likely that the amino acid is more important in the binding of the antibody to the antigen. Other methods for determining the amino acids of a variable region of an antibody that are involved in the binding of the antibody to an antigen are based on crystallographic analyzes, for example, X-ray crystallography. The term "an antibody that specifically binds to an antigen. "is proposed to include an antibody that binds to the specific antigen with significantly greater affinity than binding to any other antigen, i.e., it is proposed to define the specificity of an antibody as defined in the art. The terms "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds an antigen".

PRODUCTION OF ANTI-RECEPTOR ANTI-RECEPTOR AGENTS I. Production of Fc-receptor antibodies Anti-Fc receptor antibodies for use in the compounds that bind to the macrophages of the invention include antibodies developed using any of a number of known techniques, provided that the antibody is capable of binding to an Fc receptor in a macrophage Preferred antibodies are practical for clinical use (for example, they can be administered to humans). Particularly preferred antibodies are non-immunogenic when administered to humans (eg, they are human antibodies produced in transgenic animals), or are modified to reduce immunogenicity when administered to humans (eg, they are humanized). In one embodiment, the anti-Fc receptor antibody is a monoclonal antibody, for example, a murine or human monoclonal antibody that binds to an IgG receptor or an IgA receptor, preferably at a site that is not blocked (ie to say, linked) by a human immunoglobulin G (IgG) or immunoglobulin A (IgA). As used herein, the term "IgG receptor" refers to any of the eight genes of the Fc receptor. located on chromosome 1. These genes encode a total of 12 transmembrane isoforms or soluble of the receptor that are grouped into three classes of Fc? receptors: Fc? RI (CD64), Fc? RII (CD32), and Fc? RIII ( CD16). In a preferred embodiment, the Fc? it is a high affinity human Fc? RI. The human FcγRI is a 72 kDa molecule, which shows high affinity for monomeric igG (10 -10 M "" 1). The production and characterization of these preferred monoclonal antibodies is described by Fanger et al., In PCT Application WO 88/00052 and in the U.S. Patent No. 4,954,617, The teachings of which are incorporated herein by reference in their entirety. These antibodies bind to an epitope of Fc? RI, Fc? RII or Fc? RIII at a site that is different from the Fc-binding site? of the receptor and, thus, its binding is not substantially blocked by the physiological concentrations of IgG. The specific anti-FcγRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma that produces mAb 32 is at the disposition of the American Type Culture Collection, ATCC accession number HB9469. The anti-Fc? RI mAb 22, the F (ab'Í2) fragments of mAb 22, can be obtained from Medarex, Inc. (Annandale, NJ) The hybridoma producing mAb 22 is available from ATCC on July 9. of 1996 and has the ATCC access number assigned HB-12147. In other modalities, the anti-Fc antibody? receptor is a humanized form of monoclonal antibody 2 (H22). The production and characterization of the H22 antibody is described in Graziano, R. F. et al., (1995) J. Immunol 155 (10): - 4996-20002 and PCT / US93 / 10384. The H22 antibody that produces the cell line was deposited in the American Type Culture Collection on November 4, 1992 under the name HA022CL1 and has the accession number CRL 11177. In other embodiments, the anti-FcR antibody is specific for an IgA receptor. The term "receiver of IgA "is proposed to include the gene product of an? -gen (Fc? R) located on chromosome 19. This gene is known to encode some transmembrane isoforms alternatively spliced from 55 to 110 kDa." The Fc? R (CD89 ) is expressed constitutively in monocytes / macrophages, eosinophilic and neutrophilic granulocytes, but not in non-effector cell populations.Fc? R has medium affinity (* 5 x 10 M ") for IgAl and IgA2, which increases with exposure to cytokines such as G-CSF or GM-CSF (Morton, HC et al., (1996) Critique Reviews in Immunology 16: 423: 440). Anti-Fc monoclonal antibodies? monoclonal receptor include My 43, A 77, A62, A 59, and A3 (Monteiro et al., (1992) J. Immunol., 148: 1764; Shen et al., (1989) J. Immunol., 143: 4117). Preferred anti-FcγR antibodies are capable of binding to an FcγR without being inhibited by IgA. The A77 antibody has been produced by immunizing mice with slices of acrylamide gel containing FcγR that was affinity purified in IgA from lysates of human cells. Monoclonal antibodies were detected according to three characteristics: by staining U937 cells at a higher density after activation with PMA, selective reactivity with monocytes and blood granulocytes and their ability to immunoprecipitate molecules of approximately 55 to 75 kDa from neutrophils and U937 cells activated.

The monoclonal anti-Fc receptor antibodies used in the compounds of the invention can be produced by some techniques, including traditional monoclonal antibody methods, for example, the normal somatic cell hybridization technique of Kohler and Milstein, (1995) Nat. ? 256: 495. Although somatic cell hybridization methods are preferred, in principle, it is possible to employ other techniques to produce monoclonal antibodies, for example, viral or oncogenic transformation of B lymphocytes. A preferred animal system for preparing hybridomas is murine system. The production of hybridomas in the mouse is a well-established procedure. Immunization protocols and techniques for the isolation of splenocytes immunized for fusion are known in the art. Fusion pairs (eg, murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies (mAbs) directed against human proteins can be generated using transgenic mice that carry the entire immune system in place of the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAb with specific affinities for epitopes of a protein human (see, for example, Wood et al., International Application WO 91/00906, Kucherlapati et al., PCT Publication WO 91/10741; Lonberg et al., International Application WO 92/03918; Kay et al., Application International 92/03917; Lonberg, N. et al., 1994, Nature 368: 856-859; Green, LL et al., 1994, Nature Genet.1: 13-21; Morrison, SL et al., (1994) Proc. Nati, Acad. Sci. USA 81: 6851-6855; Bruggeman et al., (1993) Year Immunol 1: 33-40; Tuaillon et al., (1993) PNAS 90: 3720-3724; Bruggeman et al. , (1991) Eur. J. Immunol., 21: 1323-1326). In an illustrative embodiment, mice (HuMab mice) that produce a fully human antibody response after immunization can be generated by inactivating genes encoding mouse antibodies. This can be obtained by generating a "double-knockout mouse" in which the heavy chain of the endogenous immunoglobulin and the K light chain genes are broken by directed deletions of the exons coding for the constant regions (C? And JCK) . It is possible to construct separate genes containing human immunoglobulin heavy chain genes and human K light chain genes. In humans, these genes comprise approximately 1-2 megabases each, a size that is too large to isolate intact. The essential regions can be assembled in condensed form in the so-called ^ -a- & - • * - £ =. "miniloci". The heavy chain minilocus contains 2-6 segments of the V_, 15 segments of the D_ and 6 Jh gene, and the segments of the S? and C? and S? l and C? l. The light chain minilocus K contains 1-17 segments of the V ^ gene, 5 segments of the Jk gene and the Ck (Lonberg, N. et al., (1994) Na ture 368:, 856-859; Tuaillon, N et al. ., (1993) Proc. Nati, Acad. Sel. USA 90: 3720-3724). These miniloci can then be incorporated into the "double knockout" mouse genome. Some consecutive versions of these Doubleknockout / double transgenic HuMab mice can be generated, which incorporate increasing amounts of human heavy and light chain loci. For example, HuMab mice have been generated, which incorporate a 100 kb heavy chain transgene containing 6 V segments, and a 200 kb K light chain transgene containing 17 V K segments. These HuMab mice can be immunized using traditional immunization protocols, and have been shown to efficiently generate high affinity human IgGl antibodies against a broad group of antigens (Fishwild, DM et al., (1996) Nature Biotech 14: 845-851; Lonberg, N. et al. D. Huszar (1995) Int. Rev. Immunol., 13: 65-93). The antibodies generated after these protocols have been shown to have excellent biological activity and prolonged half-lives in serum.

Chimeric mouse-human monoclonal antibodies, (ie, chimeric antibodies) can be produced by known recombinant DNA techniques. For example, a gene encoding the constant region Fc of a murine monoclonal antibody molecule (or other species) is digested with restriction enzymes to separate the region encoding the murine Fc and the equivalent portion of the gene that is codes for a human Fc constant region. (See Robinson et al., Patent Application PCT International US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., Application of European Patent 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al., Et al., Patent United States No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al., (1988 Science 240: 1041-1043); Liu et al., (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. ImmunoJ. 139: 3521-3526; Sun et al., (1987) PNAS 84: 214-218; Nishimura et al., 1987, Canc. Res 47: 999-20 1005; Wood et al., (1985) Nature 314: 446-4 9; and Shaw et al., 1988, J. Nati. Cancer Inst 80: 1553-1559). The chimeric antibody can be more humanized by substituting sequences of the variable region Fv that is not directly involved in the binding to the antigen with equivalent sequences of the human Fv variable regions.

General reviews of humanized chimeric antibodies are provided in Morrison, SL 1985, Sci en 229: 1202-1207 and in Oi et al., 1986, BioTecnnigues 4: 214. These methods include the isolation, manipulation and expression of acid sequences. nucleic acids encoding all or part of the immunoglobulin Fv variable regions from at least one heavy chain or light chain. Nucleic acid sources are well known to those skilled in the art and, for example, can be obtained from 7E3, an anti-GPIIbIIIa antibody that produces hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into a suitable expression vector. Suitable humanized antibodies may otherwise be produced by substitution by CDR, US Patent 5,225,539; Jones et al., 1986 Nature 321: 552-525; Verhoeyan et al., 1988 Science 239: 153; and Beidler et al., 1988 J. Immunol. 141: 4053-4060. All CDRs of a specific human antibody can be replaced with at least a portion of a non-human CDR or only some of the CDRs can be replaced with Non-human CDRs It is only necessary to replace the number of CDRs necessary to bind the humanized antibody to the Fc receptor.

An antibody can be humanized by any method, which is capable of replacing at least a portion of a CDR of a human antibody with a CDR from a non-human antibody. Winter describes a method that can be used to prepare humanized antibodies of the present invention. (U.S. Patent Application GB 2188638A, filed March 26, 1987), the content of which is expressly incorporated by reference. Human CDRs can be replaced with non-human CDRs using site-directed mutagenesis of the oligonucleotide as described in International Application WO 94/10332, entitled "Humanized antibodies for Fc receptors for immunoglobulin G in human mononuclear phagocytes. Chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added are found in the invention In particular, preferred humanized antibodies have amino acid substitutions in the skeletal region, such as to enhance antigen binding. For example, in a humanized antibody having mouse CDR, the amino acids located in the region of the human structure can be replaced with the amino acids located in the corresponding positions in the mouse antibody.These substitutions are known to improve the binding of the antibodies humanized to antigen in some circumstances. Antibodies in which the amino acids have been added, deleted or substituted are known herein as modified antibodies or altered antibodies. The term modified antibody is also proposed to include antibodies, such as monoclonal antibodies, chimeric antibodies and humanized antibodies that have been modified by, for example, deletion, addition or substitution of antibody portions. For example, an antibody can be modified by deletion of the constant region, and replacing it with the constant region means increasing the half-life, for example, serum half-life, stability or affinity of the antibody. Any modification is within the scope of the invention as long as the compound that binds to the macrophage has at least one antigen-binding region specific for an FcR and activates at least one effector function. Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, known as the "combinatorial antibody presentation" method, has been developed to identify and isolate antibody fragments having a specific antigen specificity, and may be used to produce monoclonal antibodies (for descriptions of the combinatorial presentation of the antibody (see, for example, Sastry et al., (1989) PNAS 86: 5728; Huse et al., (1989) Science 246: 1275 and Orlandi et al. , (1989) PNAS 86: 3833) After immunization to an animal with an immunogen as already described, the antibody repertoire of the resulting B-cell combination is cloned.The methods are generally known to obtain the DNA sequence. of the variable regions of a diverse population of immunoglobulin molecules using a mixture of oligomeric initiators and PPCR, for example, the combined oligonucleotide primers corresponding to the 5 'leader sequences (signal peptide) and / or the sequences of structure 1 (FR1). ), as well as the primer for a conserved 3'-constant region primer can be used for the PCR amplification of the heavy and light chain variable regions of a number d and murine antibodies (Larrick et al., 1991, Biotec nigues 11: 152-156). It is also possible to use a similar strategy to amplify the human heavy and light chain variable regions of human antibodies (Larrick et al., 1991, Methods: Companion to Methods in Enzymology 2: 106-110). In an exemplary embodiment, RNA is isolated from B lymphocytes, e.g., peripheral blood cells, bone marrow or spleen preparations, using standard protocols (eg, U.S. Patent No. 4,683,202; Orlandi, et al., PNAS (1989) 86: 3833-3837; Sastry et al., PNAS (1989) 86: 5728 -5732; and Huse et al., (1989) Science 246: 1275-1281). The single-stranded CDNA is synthesized using primers specific for the constant region of the heavy chain (s) and each of the light chains K and γ, as well as the primers for the signal sequence. By using variable region PCR primers, the variable regions of the heavy and light chains are amplified, each alone or in combination, and ligated into suitable vectors for further manipulation in the generation of presentation packets. Oligonucleotide primers useful in the amplification protocols can be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences can also be incorporated into the primers to handle the cloning of the amplified fragment into a vector in a predetermined reading frame for expression. The V library cloned from the repertoire of antibodies from the immunization can be expressed by a population of presentation packets, preferably from the filamentous phage, to form a library of antibody presentation. In theory, the Presentation package consists of a system that allows the sampling of antibody presentation libraries with a very large diversification, rapid classification of each round of affinity separation and easy isolation of the antibody gene from the purified presentation packets. In addition to the equipment that is commercially available to generate phage display libraries (for example, Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01) and the phage display team Stratagene SurZAP catalog No. 240612). Examples of particularly manageable methods and reagents for use in the generation of a diversified antibody display library can be found in, for example, Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92/18619; Do er et al., International Publication No. WO 91/17271; Winter et al., International Publication No. WO 92/20791; Markland et al., International Publication No. WO 92/15679; Breitling et al., International Publication No. WO 93/01288; Me Cafferty et al., International Publication No. WO 92/01047; Garrard et al., International Publication No 92/09690; Ladner et al., International Publication No. WO 90/02809; Fuchs et al., (1991) Bio / Technology 9: 1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al., (1989) Science 246: 1275-1281; Griffths et al-, (1993) EMBO J 12: 725-734; Ha kins et al., (1992) J Mol Biol 226: 889-896; Claxon et al., (1991) Nature 352 = 624-628; Gram et al., (1992) PNAS 89: 3576-3580; Garrad et al., (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al., (1991) PNAS 88: 7989-7982. In certain embodiments, the domains of the V region of heavy and light chains can be expressed in the same polypeptide, linked by a flexible linker to form a single chain Fv fragment and the scFV gene subsequently cloned into the desired expression vector or phage genome. As generally described in Me Cafferty et al., Nature (1990) 348: 552-554, the complete VH and VL domains of an antibody, linked by a linker (Gly4-Ser > 3 flexible) can be used to produce an antibody single-stranded vaccine that can be converted into the separable presentation package based on antigen affinity.Smotivated scFV antibodies immunoreactive with the antigen can subsequently be formulated into pharmaceutical preparations for use in the present method.After presented on the surface of a package of In one embodiment (for example, a filamentous phage), the antibody library is detected with the FcγR, or peptide fragment thereof, to identify and isolate packets that express an antibody having specificity for FcγR.

The nucleic acid encoding the selected antibody can be recovered from the presentation package (e.g., from the phage genome) and subcloned into other expression vectors by normal recombinant DNA techniques. Anti-Fc receptor binding agents and / or other binding agents within the compounds that bind to the macrophages of the invention with high affinities for a chosen antigen (eg, surface protein) can be prepared according to the methods known in the art, for example, methods that include library detection (Ladner, RC et al., U.S. Patent No. 5,233,409; Ladner et al., RC et al., U.S. Patent No. 5,403,484). In addition, it is possible to use the methods of these libraries in detections to obtain binding determinants that are mimetic of the structural determinants of the antibodies. In particular, the Fv binding surface of a specific antibody molecule interacts with its epitope according to the principles of protein-protein interactions, hence the sequence data for VH and V_ (the latter of which may be of chain type K or?) is the basis for protein manipulation techniques known to those skilled in the art. The details of the surface of the protein that contain the binding determinants can be obtained from the sequence information of the antibodies, using a modeling procedure using three-dimensional structures previously I determined from other antibodies obtained from NMR studies or crystallographic data. See, for example, Bajorath, J. and S. Sheriff, 1996, Proteins: Struct. , Funct, and Genet. 24 (2), 152-157; Webster, D. M. and A. R. Ress, 1995, "Molecular modeling of antibody-combining sites", in S. Paul, Ed., Methods in Molecular Biol. 51, Antibody Engineering Protocols, Human Press, Totowa, NJ, pp. 17-49; and Johnson, G., Wu, T. T. and E. A. Kabat, 1995, "Seqhunt: A program to screen aligned nucleotide and amino acid sequences", in Methods in Molecular Biol 51; op. Cit., Pp 1-15. In one embodiment, the anti-Fc receptor binding agent includes an antigen-binding site that is obtained from an antibody and that is grafted onto a molecule other than the antibody. For example, it is possible to graft an antigen binding region onto a peptide or protein. In one embodiment, a portion of the antigen-binding region, For example, the portion similar to the antigen-binding region of the light chain of an antibody is grafted onto one protein or peptide and the other portion of the antigen-binding region, for example, the portion similar to the region. of antigen binding from the heavy chain of an antibody is grafted onto another protein or peptide. In In a preferred embodiment of the invention, the two proteins or peptides each having a portion of the antigen binding region are linked, for example, by chemical bonding, in recombinant form or by non-covalent interaction, such as to produce a protein that has a specific antigen-binding site for an FcR for human Ig, which activates at least one function of the effector cells mediated by the Fc receptor. An antigen-binding region can also be obtained by detecting different types of combinatorial libraries with a desired binding activity, and identifying the active species by methods that have already been described. For example, phage display techniques (Marks et al., (1992) J Biol Chem 267: 16007-16010) can be used to identify protein binding Fc? Rs. The phage display libraries have already been described. For example, a diversified peptide library can be expressed by a population of presentation packets to form a peptide display library. In theory, the presentation package consists of a system that allows the sampling of very diverse peptide presentation libraries, the rapid classification after each round of affinity separation and the easy isolation of the gene encoding the peptide from the packages of presentation purified. The peptide display libraries can be in, for example, prokaryotic organisms and viruses, which can be amplified rapidly, are relatively simple in their manipulation and which allow the creation of a large number of clones. Preferred presentation packages include, for example, vegetative bacterial cells, bacterial spores and, most preferably, bacterial viruses (especially DNA viruses). However, the present invention also contemplates the use of eukaryotic cells, including yeasts and their spores, as potential presentation packages. The phage display libraries are described in the foregoing. Other techniques include affinity chromatographies with a suitable "receptor", for example, FcγRI or FcγR, to isolate binding agents, followed by the identification of the isolated binding agents or ligands by traditional techniques (e.g. mass spectrometry and NMR). Preferably, the soluble receptor is conjugated to a tag (eg, fluorophores, colorimetric enzymes, radioisotopes or luminescent compounds) that can be detected to indicate binding to the ligand. Otherwise, it is possible to selectively release the immobilized compounds and allow them to diffuse through a membrane to interact with a receptor.

Combinatorial libraries of the compounds can also be synthesized with "tags" to encode the identity of each member of the library (see, for example, W. C. Still et al., International Publication WO 94/08051). In general, this method presents the use of inert but easily detectable labels, which are attached to the solid support or to the compounds. When an active compound is detected, the identity of the compound is determined by identification of the accompanying single label. This method of labeling allows the synthesis of large libraries of compounds that can be identified in very low concentrations among the total series of all the compounds in the library.

II. Cyanine Compositions In another embodiment of the invention, the Fc receptor binding agent of the compound that binds to the macrophage is a chemical moiety, such as a cyanine composition, including but not limited to fluorescent dye Cy5.18. OSu (known as Cy5) and conjugates and derivatives thereof. It is known that cyanine compositions bind with high affinity and specificity to the Fc? RI receptor. In certain cases, the cyanine compositions may contain two or more portions, such as a cyanin succimidinyl ester and a phycobilisome protein, for example, PE. He term "PE-Cy5" as used herein refers to the specific cascade dye composed of phycoetrine and Cy5.18.OSu; the term "PE-Cy5 reagent" designates, for example, but is not limited to, conjugates of PE-C-y5 to antibodies, to genetically engineered binding proteins and peptides (USPN 5,233,409 and 5,403,484), to avidin, to biotin, or other molecular entities. It is possible to use PE-Cy5 conjugates in therapeutic and diagnostic applications. The cyanine was isolated from the corn flower (Centaurea cyanus), and is structurally the 3, 5-diglycoside of cyanidin, which is 2- (3,4-dihydroxyphenyl) -3,5,7-trihydroxy-l chloride. -benzopyrilium and was isolated from the banana (Merck Index). Another derivative of cyanidin, 3-rhamnoglucoside isolated from bitter cherries, is described as a therapeutic application for night blindness. The anthocyanins of cranberry fruit (Vaccinium myrtillus) are marketed as nutraceutical food supplements [sic] that according to the manufacturer (Amrion, Inc., Boulder, CO), are consumed orally to improve vasodilation, decreased capillary permeability , potent collagen in blood vessels; It works as antioxidants and supports the control of inflammatory processes, improving the general vision, the stomach coatings, the blood-brain barrier and the veins of the legs and the colon (Gen. Engin, News 16 (11), p.27, 1996). The Cy5 dye derived from cyanidin, also called Cy5.18.OSu, has the chemical structure 5,5'-bis-sulfo-1, 1 '- (? -carboxyphenyl) -3,3,3', 3 '-tetramethylindodicarbocianindisuccinil ester (AS Wagoner et al., In: Clinical Flow Cytometry, p 185 (eds) A. Landay et al., The New York Academy of Sciences, New York, New York, 1993). The cyanine dye marker reagents for the sulfhydryl groups (Ernst, LA et al., 1989, Cytometry 10: 3) and the carboxymethylindycinin succinyl idyl ester (Southwick, PL et al., 1990, Cytometry 11: 418) have been described , and the compositions claimed in the patent applications (USPN 4,981,977 and 5,268,486), the contents of which are incorporated herein by reference. The structure of Cy5, and its synthesis and spectra for absorption and emission of light are given in Mujumdar, R. B., 1993, Bioconj. Chem. 4: 105. Cy5 is a sulfoindocyaninsuccinil ester, which is a cyanine dye reactive with the amino group containing a solfonated group with a negative charge on the aromatic nucleus of the indocyanine fluorophore. The Cy5 members of this family are characterized by a 5-carbon unsaturated polymethine bridge that connects two substituted ring structures. The Cy5 can be excited with a laser radiation line HeNe of 633 nm or a line of 647 nm of a laser Dr. The Cy5 and its derivatives are highlighted by photo stability, which I is comparable to or better than that of fluorescein. The extinction coefficient (L / mol cm) of 250,000 is very high. The related dyes (Mujumdar et al., Supra), with similar structures and modes of synthesis herein are encompassed within the expression "Cy5" so that this expression comprises the sulfoindocyaninsuccil esters of the cyanin dye labeling reagents in general, for example, Cy3.29.OSu (known as Cy3) and Cy7.18.OH. The terms reactive Cy5, conjugate Cy5 and derivatives Cy5 must mean a conjugate that contains at least one portion Cy5 and another molecular entity. The new additional derivatives of this basic structure already have been described, the sulfobencindocyaninsuccinil ester of the cyanine reagents (Mujumdar, SR et al., 1996, Bioconj.Chem 7: 356), which shares properties of Cy5 and other sulfoindocyaninsuccinil esters, and are contemplated to bind to Fc? RI with affinity and specificity. The use of Cy5 reagent PE-Cy5, composed of Cy5 in cascade with PE, to provide fluorescence of three colors by excitation with a single line of laser radiation of argon ion of 488 nm is described in Wagoner et al., 1993, supra, as the conditions for optimization. The Major problems with dyes this cascade based on red Texas are attributed to the instability of a portion, giving rise during the use to emission leaks in the spectrum of the other portion, limiting the ability to use the Texas network dyes that emit light close to the wavelength of this second portion. The Cy5 and its family of dye reagents, however, emit light at wavelengths larger than Texas red, so that the analysis of the data obtained from the use of Cy5 with other dyes requires minimum channel compensation in the establishment of the detection windows and in the downstream calculations. Considerations for the best mode of use of Cy5 reagents include the process of synthesizing Cy5 reagent from the components, since the ratio of the number of Cy5 molecules bound per conjugate molecule affects the spectrum at the wavelength of relative emission of the product of the synthesis. Thus, for PE-Cy5, the efficiency of the energy transfer from PE to Cy5 increases as more Cy5 molecules are bound to each PE to an optimal range, beyond which extinction interactions between Cy5 portions are observed. in excess. The optimum ratio is 4 to 8 Cy5 per PE in the PE-Cy5 cascade dye (Wagoner et al., 1993, supra), the cascade dyes are sensitive to light and their 4 Stability during use improves if the dyes are stored and handled and the experiments are conducted under dark conditions. The improved signal size due to the degree of fluorescence and absence of background for PE-Cy5, compared to the previously synthesized cascade dyes, makes it a convenient analytical tool for cell analysis studies with antibody-dye conjugates. However, at least one report of the "non-specific" binding of a variety of PE-Cy5 products from different suppliers to myeloid cells has been reported (Stewart SJ, et al., Supra), attributed to the Cy5 portion due to that the PE-Texas network conjugates do not have this property. In contrast, Takizawa et al report the binding of PE and its mAb conjugates to the low affinity mouse IgG Fc? RII and Fc? RIII receptors (J. Immunol. Methods, 1993, 162: 269).

PRODUCTION OF CYTOTOXIC AGENTS THAT KILL MACROPHATES OR REDUCE THEIR ACTIVITY I. Cytotoxins Various cytotoxic agents can be targeted to macrophages through the compounds of the invention (ie, by virtue of being bound to an agent that binds to an Fc receptor in a macrophage). As used in the present, the terms "cytotoxin" and "cytotoxic agent" include any compound (e.g., drugs) capable of killing or reducing the activity of a macrophage. For example, the compound can be a toxin, such as Gelonin, Saporin, Exotoxin A, Onconase or Ricin A, or a medicament, such as dichloromethylene diphosphonate (CL 2MDP) or a derivative thereof. The cytotoxins for use in the invention may also include an agent or a portion that improves the therapeutic activity of these compounds. For example, the cytotoxin may include an agent that promotes apoptosis, a mitotic inhibitor, an alkylating agent, an antimetabolite, a nucleic acid intercalating agent, a topoisomerase inhibitor, a specific drug of macrophages or a radionuclide. The present invention offers the advantage of targeting these cytotoxins to the Fc? of high affinity (for example, using an antibody such as Mab 22, Mab 32, or the humanized forms thereof) on macrophages where these, for example, are internalized by the cell. Therefore, these cytotoxins may be more effective in killing cells or modulating cellular function compared to other agents that are not internalized, or that are internalized with slower kinetics.

The cytotoxic agent may be a toxic drug or an enzymatically active toxin of bacterial or vegetable origin, or a fragment with biological activity ("A chain") of such a toxin. Enzymatically active toxins, 5 copies, and fragments thereof include the diphtheria A chain, the non-combining active fragments of the diphtheria toxin, exotoxin A chain (from Pseudomonas aerogunisas), ricin A chain, abrin chain A , moderation chain A, alpha-sarcin, proteins aleurites fordii, diantin proteins, American phytolac proteins (PAPI, PAPII, and PAP-S), the momordicacharantia inhibitor, curcin, crotin, the inhibitor saponaria officinalis, gelonin, mitogelin, restrictocin, fenomicin and enomicin. The preferred toxins that can be used include Gelonin, Saporin, Exotixin A, Onconase, Ricin A, diphtheria toxin and Pseudomonas exotoxin or the subunits of these toxins. Studies of the preparation, in vivo uses and pharmacokinetics of these toxins are described in, for example, Vitetta et al., (1987) Science 238: 1098-20104; Spitler, L. et al., (1987) Clin. Chem. ^ 33 (b): 1054; Uhr et al., Monoclonal Antibodies and Cancer, Academic Press, Inc. Pp. 85-98 (1983). The conjugates of the compounds of the invention and these toxic agents can be prepared using a variety of agents of coupling of bifunctional proteins as described in detail later in the section entitled "Methods of preparing conjugates of compounds that bind to macrophages". Examples of these reagents are SPDP, IT, bifunctional derivatives of imido esters such as dimethyl adipimidate, HCl, active esters such as disuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azido compounds such as bis- (p-azidobenzoyl) hexandiamma, bis-diazomo derivatives, such as bis- (p-diazonium benzoyl) ethylenediamine, diisocyanates, such as tolulene 2,6-diisocyanate [sic] and active bis-fluoride compounds, such as 1,5-difluoride-2,4-dinitrobenzene. In other modalities, the cytotoxin is a medication. Exemplary drugs include dichloromethylene diphosphonate (CL2MDP) or other chloronated derivatives [sic] (Bogers et al (1991) Clin Exp. Immunol., 86: 328-333). Otherwise, the cytotoxin may be an agent that promotes apoptosis, a mitotic inhibitor, an alkylating agent, an antimetabolite, a nucleic acid intercalating agent and a topoisomerase inhibitor. Examples of these agents that can be used in the compounds of the invention include the topoisomerase II inhibitors ellipticine, amsacrine, adriamycin and mitrozantrone, the coumarmicm prokaryotic DNA gyrase inhibitor and the DNA binding agents neocarzinostatin and chloroquine ( he - ** «" which DNA is interspersed or notched). The methods for the delivery of these medicaments, for example, the liposome supply is described below. In certain embodiments, the cytotoxin may contain a photosensitizing portion (eg, a photosensitizing medicament). The cytotoxins constituting these photosensitizing portions are useful for sensitizing an objective, for example, a macrophage, for destruction with photo activation, for example, by radiation using visible light. Preferably, the photosensitizing portion has no direct biological effect before photo activation. The compounds containing these portions can be administered to an individual, for example, topically or by injection. With photo activation by exposing these compounds to a specific wavelength of light, for example, by exposure to visible light, the portion becomes toxic (either by itself or by activating a cytotoxin associated with the portion) and selectively destroys macrophages. Without adhering to any specific theory, it is considered that the mechanism of photo activation includes transfer of energy from a photosensitizing portion to endogenous oxygen, converting it by this means into singlet oxygen. It is thought that singlet oxygen is responsible for the cytotoxic effect. The compounds that bind to the Macrophages containing photosensitizing portions are particularly useful for the treatment of dermatological diseases. Exemplary photosensitizing agents that can be used in the present invention include compounds related to porphyrin, for example, hematoporphine derivatives (Lipson, RL et al., (1961), J. Na tional Cancer Inst. 26: 1-8; Photoprine II (US 4,649,151, Dougherty, TJ (1993) Adv. Exp. Med. Bio 160 3-13, Kessel, D. et al., (1987) Photochem, Photobiol 46 463-568 and Scourides, PA et al. ., (1987) Cancer Res. 47 3439-3445), pyropheoforbide compounds (US 5,459,159; US 4,996,312, and US 4,849,207, and EP 220686), chlorophyll and bacteriophilus derivatives (EPA 93111942.4), porphycene derivatives substituted at the position 9 (WO 96/31451), frobine derivatives (WO 95/08551), as well as cloinas, phthalocyanines and porphines (reviewed in Harvey, I. Pass. (1993) J. Nati.Can.Inst. 85: 443 -457) The photo-activated forms of the photosensitizing agent, which are capable of emitting a fluorescent signal can also be used in diagnostic applications. to label compounds binding to the macrophages of the invention. In other embodiments, the compounds that bind to the macrophages of the invention may include an Fc-binding agent coupled to a therapeutic or diagnostic reagent, for example, a topical agent, through a photo-dissociable link. Preferably, the linkage is mediated by a photoactivatable agent, such as a chromophore, which releases the therapeutic or diagnostic reagent with light expression (Goldmacher et al. (1992) Bioconj Chem. 3: 104-107 ). For example, in dermatological applications, light will induce the degradation of the bond, releasing the active toxin in place (for example, the skin). Suitable photoactivatable agents for releasing the bound therapeutic or diagnostic reagent include any agent that may be attached to a functional group (eg, a phenol) or therapeutic or diagnostic reagent in which, upon exposure to light, it releases the therapeutic or diagnostic reagent in functional form. As an illustration, the photoactivatable agent can be a chromophore. Suitable chromophores are generally selected by absorption of light that can be supplied from common radiation sources (e.g., UV light in the range from 240-370 nm). Examples of photosensitive chromophores at these wavelengths include, but are not limited to, acridines, nitroaromatics and arylsulfonamides. When chromophores are used, the efficiency and wavelength at which the chromophore becomes activated photo and thus release or "discharge" the therapeutic reagent will vary depending on the functional group (s) _ £ __ particular (s) attached to the chromophore. For example, when nitroaromatics are used, such as derivatives of the o-nitrobenzyl compounds, the absorption wavelength can be significantly lengthened by the addition of the methoxy groups. In one embodiment, nitrobenzyl (NB) and nitrophenylethyl (NPE) is modified by the addition of two methoxy residues in 4,5-dimethoxy-2-nitrobenzyl (DMNB) and 1- (4,5-dimethoxy-2-nitrophenyl) ) ethyl (DMNPE), respectively, thereby increasing the absorption wavelength range at 340-360 nm (? -? ax = 355 nm). Radiation to promote photo-release of the therapeutic or diagnostic agent can be provided by a variety of sources including, but not limited to, non-coherent UV light sources and excimer sources. In one embodiment, it is possible to use a KrF excimer laser operating at 248 nanometers. Otherwise, it is possible to use a YAG laser doped with neodymium, of quadrupled frequency, in the solid state, or similar operating at 266 nm, or an argon ion laser operating at 257 or 275 nm. The photoactivatable agent can react with the therapeutic agent to create a photo-detachable bond. When chromophores are used as photoactivatable agents, the excitation wavelength can be chosen to selectively excite specific chromophores. For example, it is possible to join so that two different drugs or two different chromophores can be released with radiation to the substrate, and then independently or sequentially release the two drugs by selecting the excitation wavelength to couple the corresponding chromophores. The chromophore and the excitation wavelength can also be selected to avoid unwanted photolytic reactions of the medicament (eg, inactivation) or the surrounding tissue. For example, the photo sensitivity of nucleic acids is well known. When the drug is a nucleic acid, the excitation energy that damages the nucleic acid should be avoided (eg, wavelengths shorter than 280 nm). In addition, the compounds that bind to the macrophages of the invention can be labeled (eg, for diagnostic use) by coupling the compound to the radionuclides, such as 1311, 90Y, 105Rh, 47Sc, 67Cu, 212B? and 211At, as described in, for example, Goldenberg, D. M. et al., (1981) Cancer Res. 41: 4354-4360; in EP 0365 997; Carrasquillo et al., Cancer Treat. Rep., 68: 317-328 (1984); Zaloberg et al., J. Nati. Cancer Institute 72: 697-704 (1984); Jones et al., Int. J. Cancer 35: 715-720 (1985); Lange et al., Surgery 98: 143-150 (1985); Kaltovich et al., J. Nucí. Med. 27: 897 (1986); Order et al., Intl. J. Radxother Oncl. Biol. Phys. 8: 259-261 (1982); Courternay-Luck et al., Lancet 1: 1441-1443 (1983); Eninger et al., Cancer Treat Rep 56: 289-297 (1982); the descriptions of all of which are incorporated herein by reference. These radionuclides can also improve the cytotoxic effect of the photosensitizing portion. In these diagnostic applications, it is desirable to attach a label group to the compounds that bind to the macrophages to facilitate their detection (e.g., their binding to macrophages in a sample). Accordingly, in addition to the aforementioned radionuclides, the labeling groups include, for example, a fluorophore, a colorimetric enzyme, a radioisotope or a luminescent compound. For example, when the labeling group is an enzyme, the enzyme that binds to the macrophage binding compound will react with a suitable substrate, preferably a chromogenic substrate, in such form to produce a chemical signal that can be detected, for example, by spectrophotometric, fluorimetric or visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, peroxidase of radish, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be achieved by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the degree of enzymatic reaction of a substrate compared to standards in the same prepared manner. The detection of the binding of the compounds that bind to the macrophages with the macrophages can also be performed using any of the variety of immunoassays. For example, it is possible to use a radio immunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated in. present as reference). Otherwise, it is possible to use enzyme immunoassays (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2: 1-7, 1978, Microbiological Assayers Quarterly Publication, Walkersville, MD, Voller, et al. ., J. Clin. Phatol., 31: 507-520 (1978); # Biller, Meth., Enzymol. 73: 482-523 (1981); Maggio (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, FL.; Ishakawa, et al., (Eds.) Enzyme I munoassay, Kgaku Shoin, Tokyo, 1981). The radioactive isotope can be detected by such means How to use an accountant? or a scintillation counter or by autoradiography. It is also possible to label the compounds that bind to the macrophages with a fluorescent compound. When the fluoresced-labeled compound is exposed to light of the appropriate wavelength, it is then possible to detect its presence. Among the fluorescent marker compounds most commonly used are fluorescein, rhodamine, coerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine isothiosyanates. The compounds of the present invention can also be labeled using fluorescent emitting metals such as 152Eu, or others of the lanthanide series. These metals can be bound to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Otherwise, these compounds can be labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeling compound is then determined by detecting the luminescence that arises during the course of a chemical reaction. Examples of the particularly useful chemiluminescent labeling compounds are lummol, isoluminol, acridinium theomethyl ester, imidazole, acridinium salt and oxalate ester.

Similarly, a bioluminescent compound can be used to label the compounds that bind to the macrophages of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase, and aequorin.

CONJUGATION OF ANTI-RECEPTOR BINDING AGENTS Fc TO THE CYTOTOXINS The compounds that bind to the macrophages of the present invention contain, together with other optional components, an agent that binds to an Fc receptor in a macrophage linked to a cytotoxin. Accordingly, to produce these compounds, the anti-receptor binding agent Fc is conjugated (for example, by crosslinking covalently) to a cytotoxin using a variety of known techniques (see, for example, D. M. Kranz et al., (1981) Proc. Nati Acad. Sci. USA 78: 5807, Patent No. 4,474,893), or by recombinant expression of the anti-Fc receptor binding agent and the cytotoxin together as a fusion molecule.

Suitable agents, such as crosslinking agents, which can be employed for this purpose are well known in the art. The terms "crosslinking agent" and "crosslinker" are proposed to include molecules that can function as bridging molecules between two other molecules by means of two reactive functional groups, one of which reacts to form a covalent bond with the first molecule and the other of which it reacts to form a covalent bond with the second molecule, thereby effectively connecting the two molecules together. Preferably, the crosslinker has two reactive functional groups of different functional portions. Examples of suitable functional groups include amino groups, carboxyl groups, sulfhydryl groups and hydroxy groups. When a functional group of the crosslinker is reacted with a molecule (e.g., an Fc receptor binding agent), if necessary, it is possible to prevent the other functional group from reacting with this molecule by means of a protecting group that modifies the second functional group of the crosslinker so that it can not react with the molecule. After the first reaction is completed, the protecting group can be removed, restoring the second functional group, and then the second functional group can react with another molecule (eg, a toxin).

The compounds that bind to the macrophages of the present invention can be prepared by conjugating their constituent agents, for example, anti-FcR and cytotoxin, using methods known in the art. For example, each agent of the compound that binds to macrophages can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, it is possible to use a variety of coupling or crosslinking agents for covalent conjugation. Examples of the crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl thioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (see for example, Karpovsky et al., (1984) J. Exp. Med 160: 1686; Liu, MA et al., (1985) Proc. Nati. Acad. Sci USA 82: 8648). Other methods include those described in Paulus (Behering Ins. Mitt. (1985) No. 78, 118-132); Brennan et al., (Science (1985) 229: 81-83), and Glennie et al., (J. Immunol. (1987) 139: 2367-2375). The preferred conjugating agents are SATA ^ and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

In cases where the molecule that binds to the macrophages contains two antibodies (eg, a bispecific antibody), these antibodies can be conjugated through the sulfhydryl linkage of the hinge regions C terminal of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfur residues, preferably one, before conjugation. Otherwise, both agents can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the compound that binds to the macrophage is a mAb x mAb, mAb x Fab, Fab x F (ab'Í2 or ligand x Fab fusion protein) The compound that binds to the macrophage of the invention, For example, a bispecific molecule can be a single-stranded molecule, such as a single-chain bispecific antibody, a single-chain bispecific molecule consisting of a single-chain antigen and a binding determinant, or a single-chain bispecific molecule containing two binding determinants. Macrophage binding may also be single-stranded molecules or may consist of at least two single-stranded molecules.Methods for preparing bi-and multispecific molecules are described, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653; Patent United States No. 5,258,498; U.S. Patent No. 5,482,858. Once produced according to the above-mentioned guidelines, the compounds that bind to the macrophages can be tested for binding to macrophages using known techniques, such as the enzyme linked immunosorbent assay (ELISA), radio immunoassay (RIA), or Western blot assay. Each of these generally detects the presence of the protein-antibody complexes of specific interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an antibody or antibody-linked antibody fragment that recognizes and specifically binds to the antibody-FcR complexes. Otherwise, the complexes can be detected using any of a number of other immunoassays. For example, the antibody can be labeled in radioactive form and can be used in a radio immunoassay (RIA) (see, for example, Weintraub, B. Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). The radioactive isotope can be detected by means such as the use of a counter? or a scintillation counter or by autoradiography.

PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION The compounds that bind to the macrophages of the invention are preferably present in a composition together with a carrier or diluent. For the administration of an active m to an individual (for example, to treat or diagnose an abnormality), the compounds are preferably present with a carrier or diluent acceptable for pharmaceutical use. As described in more detail below, the pharmaceutical compositions of the present invention may be formulated especially for administration in solid or liquid form, including those accepted for the following: (1) oral administration; for example, potions (solutions or suspensions, aqueous or non-aqueous), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by intramuscular or intravenous subcutaneous injection, such as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, such as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, combined with other compounds. For example, the combination therapy may include a composition of the present invention with at least one other anti-macrophage agent, or other conventional treatment. Exemplary anti-macrophage agents include chlorinated compounds, for example, dichloromethylene diphosphonate (CL2MDP). The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a filler, diluent, excipient, liquid or solid, solvent or encapsulant material, included for transport or carry the chemical substance of an organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injuring the patient. Some examples of materials that can serve as accepted carriers for pharmaceutical use include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and waxes ~ J * "" ?? ITlii for suppositories; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; - (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) damping agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) water without pyrogens; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) solutions buffered with phosphates; and (21) other non-toxic, compatible substances that are employed in pharmaceutical formulations. A "pharmaceutically acceptable salt" refers to a salt that retains its desired biological activity of the predecessor compound. and does not impart unwanted toxic effects (see, for example, Berge, S. M. et al., (1977) J. Pharm. Sci. 66: 1-19). Examples of these salts include the acid addition salts and basic addition salts. The acid addition salts include those from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, substituted alkanic acids phenyl, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and Similar. Basic addition salts include those from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines such as N, N-dibenzylethylenediamine, N-methylglucamma, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and Similar. A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The term "administration" is intended to include any route of introduction into an individual of a compound that binds to macrophages of the invention, which allows the compound to perform its intended function (i.e., the reduction and / or inhibition of macrophages). Examples of administration routes that can be administered include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, rectal and intradermal. The pharmaceutical preparations are of course provided by convenient forms for each route of administration. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc .; administration by injection, intravenously or inhaled; topical by lotion or ointment; and rectal by suppositories. The injection can be bolus or it can be continuous intravenous.

Depending on the route of administration, the compound that binds to the macrophages may be coated with or located in a selected material to protect it from the natural conditions that may detrimentally affect its ability to perform its intended function. The compound that binds to the macrophages can be administered alone, or together with another agent as already described or with a pharmaceutically acceptable carrier or both. The compound that binds to the macrophages can be administered before the administration of another compound, simultaneously with another compound or after the administration of the other compound. In addition, the compound can also be administered in a proactive or inactive form (e.g., the compound that binds to macrophages which includes a light-sensitive toxin which becomes its active metabolite, or more active metabolite in vi). For example, with exposure to light, the phrases "parenteral administration" and "parenterally administered" as used herein means the modes of administration in addition to the enteric and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular injection. intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intraexternal and infusion. The phrases "systemic administration", "systemically administered", "peripheral administration" and "administered peripherally" as used herein means the administration of a compound that binds to macrophages, so that it enters the system of the individual and, thus, be subjected to metabolism and other similar processes, for example, subcutaneous administration. In general, the compounds that bind to the macrophages of the invention are administered locally to treat or diagnose abnormalities characterized by an abnormal number and / or function of the macrophages within a specific area or region of the body (e.g., skin). , lungs, joints or muscle / nervous tissue). For dermatological applications, the compounds are preferably delivered or administered topically or by transdermal patches. Topical administration is preferred in the treatment of skin lesions, including scalp lesions, corneal lesions (keratitis) and mucous membrane lesions where direct application is practical. Shampoo formulations are sometimes advantageous for treating leather injuries scalp such as seborrheic dermatitis and scalp psoriasis. The formulations for mouthwash and paste Oral can be advantageous for lesions in the mucous membrane, such as oral lesions and leukoplakia.

A preferred way to practice the invention is to apply the compound that binds macrophages in a carrier based on cream or oil, directly to the lesion, for example, the somatic lesion. Usually, the concentration of the compound that binds to macrophages is in a cream or oil ^ 10 is 1-2%. In addition, intradermal administration is an alternative for skin lesions such as psoriasis and wounds. Otherwise, it is possible to use aerosol topically. Oral administration is a preferred alternative for the treatment of skin lesions and other injuries described above where direct topical application is not practical, and is a preferred route for other applications. In addition, the compositions can be supplied by (parenterally, especially for the treatment of arthritis, such as sore arthritis or rheumatoid arthritis, and for direct injection of skin lesions. Parenteral therapy is usually intradermal, intra-articular, intramuscular, or intravenous. A preferred alternative is intra-articular injection in the case of treatment of 1 or only some joints (such as 2 to 6).

In addition, the therapeutic compounds are injected directly in lesions (intra-lesion administration) in appropriate cases. As an alternative in the treatment of arthritis, the compounds of the invention can be administered systemically. For the treatment of respiratory diseases, the compositions of the invention can be administered by aerosol or nasal inhalation. These compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons and / or other conventional solubilizing or dispersing agents. In certain embodiments, compositions that include the compounds can be administered systemically or locoregionally. For example, compositions of compounds that bind to macrophages that include a light sensitive portion, eg, a toxin or a linker, can be administered in this manner. In addition, some autoimmune conditions such as multiple sclerosis are preferably treated by locoregional or systemic administration of the compositions of the invention. The powders and sprays may contain, in addition to the compounds of the invention, carriers such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The Sprays can also contain the usual propellants, such as chlorofluorohydrocarbons and volatile hydrocarbons that are not substituted, such as butane and propane. Typically, an aqueous aerosol is prepared by formulating an aqueous solution or suspension of the compound together with traditional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the requirements of the specific compound, but usually include non-ionic surfactants (Tweens, Pluronics or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, solutions buffers, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions. Regardless of the selected route of administration, the compound that binds to the macrophages, which can be used in convenient hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by known traditional methods by those skilled in the art. The actual concentrations of the doses and the time of administration of the active ingredients in the pharmaceutical compositions of this invention can be varied to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a specific patient, composition and mode of administration, without being toxic to the patient. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as the controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. It is possible to use biodegradable, biocompatible polymers, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyoxyte esters and polylactic acid. Many methods for the preparation of these formulations are patented and are generally known to those skilled in the art. See, for example, Sustained and Controlled Relay Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc. New York, 1978. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. . For example, the compound can be administered to an individual to a suitable carrier, for example, liposomes, or a diluent. Acceptable diluents for pharmaceutical use include saline and buffered aqueous solutions. Liposomes include water-in-oil-in-water CGF emulsions, as well as traditional liposomes (Strejan et al., (1984) J. Neuroimmunol.1: 27). Acceptable carriers for pharmaceutical use include sterile aqueous solutions or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of this medium and agents for pharmaceutically active substances is known in the art. Except for any traditional medium or agent that is incompatible with the active compound, use thereof is contemplated in the pharmaceutical compositions of the invention. The complementary active compounds can also be incorporated into the compositions. Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, micremulsion, liposome or other convenient structure suitable for the high concentration of the medicament. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and convenient mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the necessary particle size in the case of dispersion and by the use of surfactants. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be obtained by including in the composition an agent that retards absorption, for example, salts of monostearate and gelatin. Sterile solutions can be prepared by incorporating the active compound in the amount necessary in ^ 10 a suitable solvent with one or a combination of aforementioned ingredients, as required, followed by microfiltration for sterilization. In general, the dispersions are prepared by incorporating the active compound in a sterile vehicle containing a dispersion medium. basic and the other necessary ingredients of the aforementioned. In the case of sterile powders for the preparation of sterile injectable solutions, the methods Preferred preparation methods are vacuum drying and freeze drying (lyophilization) which produces a powder of the Active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. Dosages are adjusted to provide the desired optimal response (for example, a response therapeutic). For example, it is possible to administer a single bolus, several divided doses may be administered during the time or the dose may be reduced or increased proportionally as indicated by the exigencies of the therapeutic situation. It is especially convenient to formulate the compositions in dosage unit forms to facilitate administration and uniformity of dosage. The unit form of the dose as used herein refers to physically small units suitable as unitary dosages for the individuals to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated and directly depends on: (a) the unique characteristics of the active compound and the specific therapeutic effect to be obtained, and (b) the limitations inherent in the technique of composition as it can be an active compound for the treatment of sensitivity in individuals. Examples of antioxidants acceptable for pharmaceutical use include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hiroxianisol (BPA), hydroxytoluene butylated (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as acid citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. For the therapeutic compositions, the formulations of the present invention include those which are suitable for topical, dermal or epidermal administration. The formulations can conveniently be presented in unit dosage forms and can be prepared by ^ P 10 any of the methods known in the art of pharmacy. The amount of the active ingredient that can be combined with a carrier material to produce an individual dosage form will vary depending on the individual being treated and the specific mode of administration. The The amount of the active ingredient that can be combined with a carrier material to produce an individual dosage form will generally be that amount ^ of the composition that produces a therapeutic effect. In general, 100%, this amount will range from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%. Dosage forms for topical administration or transdermal of the compositions of this invention They include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with the pharmaceutically acceptable carrier, and with any of the preservatives, buffers or propellants that may be necessary. Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the invention include: water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and convenient mixtures thereof, vegetable oils like olive oil, and injectable organic esters such as ethyl oleate. It is possible to maintain adequate fluidity, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents or, emulsifying agents and dispersing agents. The prevention of the presence of microorganisms can be guaranteed by the sterilization procedures, supra, and by the inclusion of some antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable include ostonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be obtained by including agents that delay absorption such as aluminum monostearate and gelatin. When the compounds of the present invention are administered as pharmaceutical forms, to humans and animals, they may be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (most preferably, 0.1 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier. The concentrations of the actual dose of the active ingredients in the pharmaceutical compositions of this invention can be varied to obtain an amount of the active ingredient which is effective to obtain the desired therapeutic response for a specific patient, the composition and mode of administration, without being toxic to the patient. The concentration of the selected dose will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the speed of excretion of the particular compound that is used, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions that are employed, the age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in the medical art. A doctor or veterinarian with ordinary skills can easily determine and prescribe the effective amount of the necessary pharmaceutical composition. For example, the doctor or veterinarian can establish the doses of the compounds of the invention used in the pharmaceutical composition in concentrations lower than necessary to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is had. In general, the convenient daily dose of a composition of the invention will be that amount of the compound that is the dose. lowest effective to produce the therapeutic effect. This effective dose will generally depend on the factors already described. It is preferred that the administration be local, for example, topical, subcutaneous, intradermal, preferably administered close to the target site. If desired, the effective daily dose of the therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at suitable intervals during the day, optionally, in unit dosage forms. Although it is possible to administer only one compound of the present invention, it is preferred to administer the compound as a pharmaceutical formulation (composition). The therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices described in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses a micro-infusion pump, which can be implanted to dose the medication at a controlled rate; U.S. Patent No. 4,486,194 which describes a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233 which describes a pump for administering drugs intravenously to deliver the drugs at a precise rate; U.S. Patent No. 4,447,224 which describes an infusion apparatus that can be implanted for variable flow for continuous delivery of the medicament; U.S. Patent No. 4,439,196 which describes an osmotic system for the delivery of medicaments having multi-chamber compartments; and U.S. Patent No. 4,475,196 which describes an osmotic system for the delivery of medicaments. These patents are incorporated herein by reference. Many other of these implants, delivery systems and modules are known to those skilled in the art. In certain embodiments, the compounds of the invention can be formulated to ensure adequate distribution in vivo. In one embodiment, the molecules that bind to the macrophages can be encapsulated in liposomes. For the methods of manufacturing the limposomes, see, for example, U.S. Patent 4,522,811; 5,374,548; and 5,399,331. The liposomes can consist of one or more portions that are selectively transported to specific cells or organs, thus ensuring the targeted delivery of the drug (see, for example, V. V. Ranade (1989) J. CJin Pharmacol. 29: 685). For example, in certain embodiments it is preferable to use single chain antibodies against an Fc receptor (scFv), for example, H22 scFv, to direct the compounds of the invention to the Fc-bearing macrophages. Protocols for preparing the scFv fragments encapsulated in liposomes are described in Kruif, J. et al., (1996) FEBS 399: 232-236. For example, the H22 scFv modified with lipids may be coupled to the liposomes composed of egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), cholesterol and, optionally, rhodamine-phosphatidylethanolamine (rhodamine) as a fluorescent bilayer marker, in a molar ratio of 10: 1: 5: 0.01, diluting mixed micelles containing n -octyl? -D-glucoside, lipids and scFv modified with lipids at a concentration well below the critical concentration of the detergent levels. The incorporation of the scFv molecules into the liposomes can be verified by SDS-PAGE. A "therapeutically effective dose" is that dose which reduces the number of macrophages within a selected treatment area relative to an untreated control, or which inhibits the activity of the macrophages within a selected area so that, for example, these no longer proliferate or contribute inflammatory responses within the area. As a consequence, the symptoms of the disease mediated by macrophages are improved. The ability of the compounds of the invention to kill or inhibit a population of macrophages can be evaluated in an animal model system, such as a transgenic animal expressing a human Fc receptor as described in the examples herein. Otherwise, these functions can be evaluated in known in vi tro trials for expert practitioners. A therapeutically effective amount of a therapeutic compound may decrease the population or activity of the macrophage cells, or otherwise lessen the symptoms in an individual. A person skilled in the art will be able to determine such amounts based on factors such as the size of the individual, the severity of the symptoms of the individual and the particular composition or route of administration selected. The composition must be sterile and fluid to the extent that the composition can be delivered by syringe.

V 10 In addition to water, the carrier can be a buffered, isotonic saline solution, ethanol, polyol (for example, glycerol, polyethylene glycol and liquid polyethylene glycol and the like), and convenient mixtures thereof. Proper fluidity can be maintained, for example, by using coating as lecithin, by maintaining the necessary particle size in the case of dispersion and by the use of surfactants. In many cases, it is It is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol and sodium in the composition. The long-term absorption of the injectable compositions can be achieved by including in the composition an agent that retards absorption, for example, aluminum monostearate or gelatin.

USES AND METHODS OF THE INVENTION The compounds that bind to the macrophages of the? present invention have some diagnostic, therapeutic and research utilities. These can be administered to cells in vi tro (in culture), ex vi ve or in vivo (in an individual), to treat, diagnose or study a variety of anomalies. In one embodiment, a method is provided for depleting (e.g., reducing the number) or inhibiting the activity of I 10 macrophages in a selected treatment or diagnostic area. The method includes contacting the selected area with the compound that binds to the macrophages in an amount sufficient to obtain the aforementioned result. As used herein, the terms "selected area" or "local area" refers collectively to any tissue sample or selected cell (in vi tro or in vivo) that contains, or may contain, macrophages that contribute to an anomaly, such as a localized area of the human body (skin, 20 lungs, joints, etc.) or a sample of tissue culture. Contact can occur in vi tro (for example, cells in culture) or in vivo (for example, by administering the compounds of the invention to an individual).

As used herein, the term "individual" is intended to include human and non-human animals. Preferred human animals include a human patient having an abnormality characterized by aberrant activity of a macrophage cell, for example, a macrophage cell of the skin. The term "activity" is proposed to include all the biological functions of a macrophage cell, including its proliferation, differentiation, survival, growth factor or cytokine secretion, among others. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like. The compounds that bind to the macrophages of the invention can initially be tested in vi tro. For example, the activity of these molecules that kill and / or modulate, for example, by reducing, the activity of the macrophage can be tested on cell lines from macrophages, differentiated blood monocytes, cultured and primary culture systems. Protocols for testing the activity of the compounds that bind macrophages can be found, for example, in Immunopharmacology of Macrophages and Other Antigen-presenting Cells (ISBN 0-12-137800-4, Academic Press Limited ). For example, cultures of skin macrophages. primary, can be established from skin cells from healthy and dermatological individuals [sic]. The activity of macrophages, for example, cell proliferation or secretion of cytokines can be tested at specific intervals after the addition of a series of concentrations of the compounds of the present invention. In one modality, it is possible to use "biopsies" obtained from healthy and dermatological individuals. Puncture biopsies can be cultured submerged or with the epidermal side on the surface of the culture medium, to which the compounds of the invention can be added. After cultivation with the compounds that bind to the macrophages of the invention, it is possible to test the effect (s) of these compounds on the activity of the macrophages by immunohistochemistry or by ELISA, RIA or EIA. Protocols for detecting changes in cell proliferation, e.g., thymidine or BrdU incorporation assays, are known in the art. The compounds that bind to the preferred macrophages of the invention decrease or eliminate the activity of the macrophages. Protocols for detecting changes in cytokine concentration can be detected through a variety of immunoassays such as enzyme linked immunosorbent assay (ELISA), enzyme immunoassay (EIA) or radio immunoassay 7 (RIA) which are known in the art (see, for example, Keler, T. et al., (1997) Cancer Research 57: 4008-14). Exemplary cytokines that can be tested include: granulocyte / macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), interleukins 1-12 (IL-1 to IL-12), and TNF- ?. The concentration of a cytokine can be measured using EIA by detecting the interaction of the cytokines with an antibody, which in turn is conjugated to an enzyme. The activity of the enzyme is detected by reaction with a suitable substrate, preferably a chromogenic substrate, in such form to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or visual means (Voller, The Enzyme Linked Immunosorbent Assay (ELISA), "Diagnostic Horizons 2: 1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller, et al., J. Clin. Pathol., 31: 507-520 (1978); Butler, Meth Enzymol 73: 482-523 (1981); Maggio, ed.) Enzyme Immunoassay, CRC Press, Boca Raton, FL., 1980; Ishikawa, et al., (Eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981).

Enzymes can be used to detectably label the antibody as described above. Detection can be carried out by colorimetric methods that employ a chromogenic substrate for the enzyme. Detection can also be carried out by visual comparison of the degree of the enzymatic reaction of a substrate compared to similarly prepared standards. The detection of a cytokine can also be achieved using a radio immunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated in the present as a reference). The radioactive isotope can be detected by means such as the use of a counter? or by a scintillation counter or by autoradiography. It is also possible to label the anti-cytokine antibody with a fluorescent compound. When the antibody labeled in fluorescent form is exposed to light of adequate wavelength, its presence can then be detected. Among the most commonly used fluorescent marker compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorine scamin. The antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be bound to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or acid ethylenediaminetetraacetic (EDTA). The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the antibody labeled with chemiluminescence is then determined by detecting the luminescence that arises during the course of the chemical reaction. Examples of the particularly useful chemiluminescent labeling compounds are luminol, isoluminol, acrominic acridinium ester, imidazole, the acridinium salt and the oxalate ester. In the same way, it is possible to use a bioluminescent compound to label the antibody. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. The important biolucent compounds for labeling purposes are luciferin, luciferase and aequorin. Compounds that bind to macrophages can also be tested in vivo. For example, these compounds can be treated using mice expressing human Fc receptors as described in the examples herein. In one embodiment, the compounds that bind to the macrophages can be injected intradermally in these transgenic mice. The controls injected with vehicle can be processed in parallel. Chronic skin inflammation in these mice can be experimentally induced by repeated topical application of 5% sodium lauryl sulfate. The effects of these compounds can be monitored immunohistochemically, for example macroscopically or clinically, at different times after injection. The compounds that bind to the macrophages of the invention can be used in the treatment of abnormalities characterized by activity or aberrant numbers of macrophages. The term "aberrant" refers to a density of macrophages within a selected site that is different (for example greater) than that found in the same area in normal, healthy patients. The term "aberrant" also includes abnormal activity of macrophages, such as abnormally high cell proliferation or cytokine secretion. Accordingly, in one embodiment, the invention provides a method of prophylactic treatment or prevention of conditions characterized by numbers or aberrant activities of macrophages in a selected area, which consists of administering to an individual, generally in the local area in need of treatment. , a pharmaceutical composition containing one or more compounds that bind to macrophages.

Compounds that bind to macrophages are generally used as targeting agents to deliver cytotoxins (eg, drugs) to receptor-carrying macrophages (Fc). In one embodiment of the invention, the cytotoxin is encapsulated within a liposome that by itself is directed to the macrophages carrying the Fc receptor. Thus, the compound that binds to the macrophages contains an antireceptor binding portion Fc linked to a liposome containing a cytotoxin. In a preferred embodiment, the anti-Fc receptor binding portion is a single-chain antibody directed against an Fc receptor (scFv), such as H22 scFv. The anti-FcR scFv is ligated or inserted into the lipid bilayers of the liposome in a form that allows the scFv to still recognize and bind to the Fc receptors outside the liposome. This can be done using known protocols, such as those described by Kruif, J. et al., (1996) FEBS 399: 232-236. The final result is an FcR-oriented cytotoxin that is delivered to the cells in the form of a liposome. As used herein, a "therapeutically effective amount" of a compound that binds to macrophages refers to an amount of a compound that is effective, with the administration of a single dose or multiple doses to the individual, inhibiting growth of the cells or an improvement in clinical symptoms in the absence of such treatment. As used herein, "a prophylactically effective amount" of a compound refers to an amount of a compound that binds to macrophages that is effective, with a single or multiple doses administered to the patient, in the prevention or delay of the presence of the onset or recurrence of a disease state mediated by macrophages. The terms "induces", "inhibits", "potency", "elevates", "increases", "decrements" or similar, for example, denoting quantitative differences between two states, refers to the differences that are at least statistically significant among the two states. For example, an "amount effective to inhibit the growth of macrophage cells" means that the growth rate of the cells will differ in a least statistically significant way from the untreated cells.

The compounds that bind to the macrophages of the invention can be used to treat a variety of diseases mediated by macrophages. These diseases are not necessarily characterized by the numbers and / or aberrant activity of the macrophages, but each may include undesired activity of the macrophages that is detrimental to the patients. In one embodiment, the compounds are used to treat autoimmune diseases including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, soriatic arthritis, multiple sclerosis, encephalomyelitis, diabetes, myasthema gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis ( including atopic dermatitis and eczematous dermatitis) soasitis, Sjogren, including keratoconjunctivitis sicca secondary to Sjogren syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus eosthetosus, scleroderma, vaginitis, proctitis, drug rash, reverse lepromatous reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing haemorrhagic encephalopathy, senso-neural hearing loss, progressive, bilateral, idiopathic, aplastic anemia, pure erythrocyte anemia Idiopathic thrombocytopenia, polycontis, Wegener's granulomatocyst, chronic active hepatitis, Stevens-Johnson syndrome, sprue, lichen planus, Crohn's disease, Graves' ophthalmopathy, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial lung fibrosis) . The decreasing modulation of the immune activity It will also be desirable in cases of allergy such as atopic allergy. (B Examples of preferred autoimmune / dermatological abnormalities for which the method can be used as part of a treatment regimen include: psoriasis, atopic dermatitis, multiple sclerosis, scleroderma and cutaneous lupus erythematosus. For example, the methods and compositions of the invention can be used to treat atopic dermatitis (AD). Without sticking to the theory, it ^ P 10 considers that during the acute phase of cutaneous inflammation in AD, the phenotype of local T cells is exchanged from an initial Th2 type to a Thl type in the chronic phase. At this point, an increase in the production of IL-12 in the lesion is found, along with a strong influx of activated inflammatory macrophages. Macrophages are potent producers of IL-12 that induces T cells to produce IFN-α, which in turn is a potent activator of macrophages (Thepen, T. et al., (1996) j Allergy Clin. Immunol. 97: 828-837; Grewe, M. et al., (1998) Immunol. Today 19: 359361). This positive feedback potentially creates a vicious circle, which by itself may be able to maintain local inflammation without the need for external stimuli. Other mechanisms, which result in a non-specific response of the allergen, continuous, resulting from the deregulation of the macrophages they are plausible, considering the regulatory potential of macrophages. The selective and localized elimination of? Inflammatory macrophages by targeting an Fc receptor, eg, FcγRI, described in the following Examples 5 makes the compositions of the invention useful in reducing or eliminating the positive feedback cycle created in the secretion of macrophages, and by treating so diseases like AD. Additional examples of diseases that can be treated by the therapeutic methods of the invention include infectious diseases, for example, HIV infections, respiratory states such as mild polymorphic dermatosis (CPLD), chronic obstructive pulmonary diseases (COPD), for example. allergic asthma and 15 sarcoidosis, and inflammatory reactions such as those seen in open wounds and burn wounds. In other embodiments, the compositions and methods of the present invention can be used in cosmetic applications. For example, the compounds that bind to the macrophages can be applied locally (for example topical) to the skin to delay and / or prevent the aging process of the skin. The therapeutic methods of the present invention can be carried out together with other techniques for elimination of the macrophage cells. For example, the treatment using the compounds that bind to the macrophages of the invention can be used in conjunction with surgery, chemotherapy or radiotherapy. The compounds that bind to the macrophages of the invention can also be used to modulate the concentrations of FcγR on the effector cells, such as by coronation and elimination of the receptors on the cell surface. For this purpose it is also possible to use mixtures of the Fc anti-receptors. The present invention further provides a kit containing one or more doses of a compound that binds macrophages and instructions for its use. In other embodiments, the combinations of the macrophage binding compounds of the invention can be used to selectively kill or reduce the activity of the macrophages, for example, a combination of a first compound having at least one specific antigen binding region. for FcR and a toxin, and a second compound having an antigen binding region for an epitope different from the FcR receptor or a different Fc receptor, for example, an Fc ?. In certain embodiments, it is possible to use a second compound that binds to the macrophages of the invention together with the first. For example, this second compound that binds to Macrophages can have at least one specific antigen-binding region for an IgA receptor, for example, an Fc receptor, an IgE receptor, for example, the Fc receptor, an Fc receptor. and / or an Fc ?. Before administering the compounds that bind macrophages to an individual, the individual can be pretreated with a compound that modulates, for example, that enhances or inhibits the expression or activity of the Fc ?, receptors by, for example, the treatment the individual with a cytokine. Preferred cytokines for administration during treatment with the compound that binds to macrophages include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon- ? (IFN-?), And tumor necrosis factor (TNF). The compounds that bind to the macrophages of the invention can also be used for in vitro and in vivo diagnosis to detect and / or measure populations of macrophages by measuring the concentrations of the Fc receptor binding. For example, as shown in? In the examples provided herein, abundant expression of FcγRI is detected in the dermis of acute and chronic cutaneous inflammation in humans. Therefore, the compounds that bind to the macrophages and which are described herein can be used to diagnose these states inflammatory For such uses, the compound can be linked to a molecule that can be detected. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme, or a coenzyme cofactor. Accordingly, in another embodiment, the invention provides a method for diagnosing in vi tro or in vi ve conditions characterized by aberrant numbers of macrophages (e.g., proliferation of macrophages) and / or expression of the Fc receptor (e.g. an increased number of cells expressing an Fc receptor and / or increased expression of the Fc receptor in a given cell). By measuring the level of binding of the compounds of the invention in a given test sample or within a localized area, it is possible to deduce the presence of the macrophages within the area or sample, provided that the anti-Fc receptor component of the compound is specific for the Fc receptors of macrophages. This can be done: (i) by obtaining a body sample, such as a body fluid, tissue (eg, a skin sample) or biopsy, a patient; (ii) contacting the body sample with a compound that binds to the macrophages of the invention or a fragment thereof; (iii) determining the level of binding of the compound that binds the macrophages to the body sample; (iv) compare the amount of the molecule bound to the sample body with a control sample, for example, a biological sample from a healthy individual, or with a predetermined level?) base, so that a union greater than the control level is indicative of the presence of a disease by macrophages, for example, skin disease. Preferably, the expression level of the Fc receptor is detected mainly in the population of macrophage cells relative to other cells expressing the Fc receptor. The protocols for diagnostic tests? 10 in vivo and in vi tro are provided in PCT / US88 / 01941, EP 0 365 997 and US 4,954,617. The following invention is further illustrated by the following examples, which should not be considered as limiting. The contents of all references, 15 pending patent applications and published patents mentioned in the application are expressly incorporated by reference herein.

EXAMPLES 20 Materials and methods The following methods were used in the studies described below. The compound terms that bind to macrophages, immunotoxins CD64 (CD64IT) or Immunotoxins (IT) are used interchangeably in the present.

Monoclonal Antibodies The following examples describe the use of an anti-CD64 (anti-FcR) antibody corresponding to a humanized form of monoclonal antibody 22 (H22), described in USPN 5,635,600, which is incorporated by reference. The production and characterization of the H22 antibody is described in Graziano, R. F. et al., (1995) J. Immunol 155 (10): 4995-5002 and PCT / US93 / 10384. The cell line product of the H22 antibodies was deposited in the American Type Culture Collection on November 4, 1992, under the designation HA022CL1 and has the access number ATCC CRL 11,177. Other specific anti-CD64 antibodies that can be used in the methods and compositions of the invention are Mu? No mAb 32.2, mAb 44, mAb 62 and mAb 197 antibodies. The hybridoma that produces mAb 32.2 is available from the American Type culture Collection access number ATCC HB9469. The preparation of the conjugates mAb 197-R? Cm A is described in the following examples. The anti-FcR mAbs conjugates were purified from each respective hybridoma supernatant by affinity chromatography with protein A (Bio-Rad, Richmond, CA).

Immunohistochemical staining Stain A: CD64 Biopsies were cut into sections of 6 microns in a microtome by freezing and mounted on covered slides. After drying overnight, the sections were fixed for 10 minutes with anhydrous acetone and air dried. The slides were incubated with conjugated FITC 10.0 (Serotec 1:40) in PBS 2% mouse normal serum (NMS) for 45 minutes. The slides were washed three times for 5 minutes with PBS, 0.05% Tween, after which alkaline phosphatase (AP) conjugated sheep anti-FITC (Boehringer Mannheim, 1: 400) in PBS (1% human AB serum, 1% NM? For 30 minutes). After washing twice in PBS / Tween and once in Tris-HCl (0.1 M, pH 8.5), PA activity was demonstrated using naphthol AS-BI phosphate (the sodium salt, 50 mg / 100 ml; Sigma) as substrate and fuchsin (10 mg / 100 ml; Merck, Whitehouse Station, N.J.) new as chromogen dissolved in 0.1 M Tris HCl, pH 8.5, giving rise to a red-pink stain. The activity of the endogenous PA was inhibited by the addition of levamisole (34 mg / 100 ml, Sigma) to the reaction mixture. The slides were slightly stained with hematoxylin.

B: Markers f Sections were fixed in anhydrous acetone with H2O2 (30%, 100 μl / 100 ml) for 7 minutes. Slides 5 were incubated with primary rat antibodies at optimal dilution for 45 minutes in PBS 2% NMS. The following antibodies were used as staining macrophages: MOMA-2 (Kraal, G. et al., (1987) Scand, J. Immunol., 26: 653-661); Dendritic cells: NLDC145 (Kraal, G. et al, (1986) J. Exp. Med. 163: 981-997); T cells: KT3 (Tomonari, K. (1988) Immu? ogenetics 28: 455-458). After washing three times (5 minutes in PBS, 0.05% Tween 20), incubation with rabbit anti-rat peroxidase-labeled conjugate (DAKO, 1: 200), in PBS (1% human AB serum, 1% NMS) followed during 30 minutes. After rinsing twice with PBS and once with NaAc (0.1 M, pH 5.0), PO activity was revealed using H2O2 as substrate and DAB (Sigma) as chromogen, causing a brown stain.

Animal studies Induction of cutaneous inflammation, immunotoxin injections and biopsies. In the experiments described herein, transgenic FVB / N mice expressing human Fc? RI (Heijnen, I. A. and col., (1996) J. Clinlnvest. 97: 331-338). Baits not transgenic served as controls. To induce chronic cutaneous inflammation, an area of 1.5 x 1.0 cm in both ^ flanks of the mice were shaved and sodium lauryl sulfate (SLS) (5% in saline) irritant, epicutaneous was applied daily 10 consecutive days. The animals were anesthetized with 20 μl of a 4: 3 mixture of Aescoker (Aesculaap, Gent, Belgium) and Rompun (Bayer, Leverkusen, Germany), injected intramuscularly. Two adjacent intradermal injections, (10 μl each, 2 x P 10 10 M), in relation to the Ricin-A portion, in saline) were administered. For control purposes, identical saline injections were collaterally administered. The animals were anesthetized as described above and biopsies were taken by 3 mm puncture, frozen in instantaneously in liquid nitrogen and were stored at -70 ° C before use. The skin was closed with a suture. The puncture biopsies (3 mm) were taken with local anesthesia (1% lidocaine) from the lesioned AD skin (n = 3), 24 h APT (n = 3), 48 h SLS (n = 2) and 72 h WB challenged the PLE skin. The biopsies were frozen in liquid nitrogen and stored at -70 ° C before use.

EXAMPLE 1: PREPARATION OF IMMUNOTOXINS CD64 Monoclonal antibodies CD64 197 (Guyre, P.M. and 25 col., 1989, J. Immunol. 143: 1650-1655 and H22 (Graziano, R.

F. et al., 1995 J. Immunol. 155: 4995-5002) were conjugated to deglycosylated Ricin-A (30 KDa, Sigma) I using a convenient linker (such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (Pierce) 5 heterobifunctional dissociable crosslinker Under conditions GLP according to the manufacturer's instructions In short, the SPDP was conjugated to the CD64 mAb, for example, H22, then the molar ratio of mAb-PDP was determined.After determining the molar ratio of mAb-PDP, added Ricin A. Free PDP groups and free Ricin A chains were inactivated and the mixture was purified by size exclusion chromatography. The purity of the H22-Rici-A conjugates was also checked by SDS-PAGE. The H22-Ricin-A conjugates were sterilized using a filter of 0.2 ?. All steps of the preparation were carried out under conditions of Good Manufacturing Practices.

EXAMPLE II: EFFECTIVE CELLULAR DEATH OF MACROPHATES 20 USING IMMUNOTOXINS CD64 0 Constitutive expression of FcγRI is limited mainly to cells of the myeloid lineage, and is strongly activated under proinflammatory and inflammatory conditions (Velde, AA et al., (1992) J. Immunol 149: 4048-4052; Schiff, D. E. et al., (1997) Blood 90: 3187- 3194). The ability of Fc? RI to rapidly and efficiently mediate endocytosis makes this receptor an effective target for? activated inflammatory macrophages (Heijnen, I. A. et al., (1996) J. Clin. Invest. 97: 331-338). Some imunotoxins 5 against hFc? RI were prepared as described in Example 1 using conjugates of Ricin-A toxin and CD64 antibodies. To establish the effectiveness of these conjugates in the induction of death of the macrophages, the line of '10 human promonocytic cells, cultured U937, not stimulated or stimulated with IFN- ?, was examined in the presence or absence of the compositions of the present invention. invention (Figures 1, A and B). The culture conditions and the stimulation of U937 cells with cytokines are described in Guyre, P. M. et al., (1983) J. Clin. Invest. 72: 393-397. Briefly, U937 cells were cultured in the presence of 300 U / ml of IFN? for 24 hours to activate the expression of Fc? RI. The levels of Fc? RI were monitored by flow cytometry. In addition, cells IIA1.6, not transfected or transfected with cD ^ lA for Fc? RIa were tested. The IIA1.6 cells were obtained from murine A20 B cell lymphoma and were recently shown to belong to a subset other than B cells / macrophage CD5 + cells (Van Vugt, M. J. et al., 1998. Clin Exp.

Immunol. 113: 414-422).

The cytotoxic efficacy of CD64 immunotoxin (IT) was assessed by measuring the inhibition of [H] thymidine incorporation in a concentration-dependent mode (Post, J. et al., Leuk Res. 19: 241-247). Briefly, the cells were seeded at 5 x 10 cells / well in a 96-well round base plate and incubated with CD64 IT for 72-12 hours at concentrations ranging from 10 to 10"M with reference to the ricin portion. The cells were pulsed for 4 hours with [H] thymidine (l? Ci) and subsequently harvested in glass wool filters and counted in a beta-plate scanner.All incubations were performed in culture medium supplemented with human AB serum. to 2% to block the Fc binding site of Fc? RI, thereby allowing the binding of IT through its unique antigen recognition site.The number of cells seeded was chosen so that the incorporation of [H] thymidine was a linear function of the cell number The background values of the [H] thymidine incorporation were obtained by incubation with 0.1 mM cycloheximide, the results were expressed as a percentage of [H] -thymid incorporation ina compared to cells treated in the same way for control. In Figures 1A-1B, the bar graphs represent the percentage of incorporation of [H] -thymidine compared to that of the control with medium (± SEM). The dose-dependent decrease in the incorporation of [H] -thymidine as a function of the increasing concentrations of H22-R or 197-R show the cytotoxicity of the immunotoxins in the stimulated U937 cells. For panels 1C-1D, the bar graphs represent the percentage of incorporation of [H] -t? Midin compared to that of the control of the medium (± SEM). The dose-dependent decrease in the incorporation of [H] -thymidine with respect to increasing concentrations of H22-R or 197-R show the cytotoxicity of immunotoxins in IIA1.6 cells transfected with hFc? -RI. This demonstrates the specificity of both ITs for cells expressing hFc? RI. The two immunotoxins tested were potent inducers of cell death. However, H22 Ricin-A (H22-R) was especially more effective than 197 Ricin-A (197-R) in inducing cell death, especially in non-stimulated -8 -9 cells. Incubation with Ricin-A only at 10 and 10 M had no significant effect (88.9 ± 14.2 and 100.4 ± 13.5 percent, respectively). In addition, no significant effect of IT was found on the non-transfected IIA1.6 cells, contrary to the efficient annihilation of the IIA 1.6 cells transfected with hFc? RI detected using any of these IT (Figure 1, panels C and D). These results demonstrate the efficacy and specificity of CD64 IT in the death of cells expressing hFc? RI in vi tro. Based on these experiments, H22-R was used at a concentration of 2 x 10"M in the experiments in vi, which are described below.

EXAMPLE III: INDUCTION OF APOPTOSIS BY IMMUNOTOXINS CD64 To establish whether the cytotoxic effect of H22 Ricin-A was due to induction of apoptosis, staining with propidium iodide in hypotonic buffer was performed. In this test the segmented apoptotic nuclei are recognized by the I 10 content of the subdiploid DNA. To perform these experiments, nuclear segmentation was detected using propidium iodide staining as described in Nicoletti, I., et al., (1991) J. Immunol. Methods 139: 271-279. In brief, the cells were incubated with IT and harvested at different times. Cells were fixed with ethanol at -20 ° C, incubated with extraction buffer (0.05M Na2HP04, 0.0025M citric acid, 0.1% Triton X-100, 20μg / ml propidium iodide). The fluorescence of propidium iodide was analyzed using a flow cytometer Fluorescent Activated Cell Sorter (FACScan) __, (Beckton and Dickinson, San Jose, CA) As shown in Figure 2, apoptotic nuclei were detected in cultures treated with IT in relation to a control. In this experiment, the U937 cells were stimulated with IFN? and incubated for six hours with different concentrations of H22-R. Apoptotic nuclei were detected during the first two hours after IT exposure and were still evident after 16 hours of treatment. These findings show that the Cytotoxic effect of H22 Ricin-A IT results from the induction of apoptosis. Cell death mediated by apoptosis limits potential detrimental effects by depleting cells expressing hFc? RI in vi. In addition, long-lasting cell death induced by H2-R (even after 16 hours) suggests the practicability of H22-R as IT for the depletion of cells expressing hFc? RI in vi vo.

EXAMPLE IV: DETECTION OF CELLS EXPRESSING Fc? RI IN CHRONIC CHRONIC INFLAMMATION IN HUMANS 15 The staining capacity of another monoclonal antibody CD64, 10.1 (Dougherty, GJ et al., 1987 Eur. J. Immunol. 17: 1453-1459 ), was tested after preincubation of the sections with H22 antibodies and in the presence of different concentrations of the H22 antibody. Since the 10.1- and H22 recognize different epitopes in hFc? RI, no significant change in intensity of staining or pattern was detected with simultaneous incubation. Based on these results, antibody 10.1 was used in all the experiments that included the immunohistochemical evaluation of the tissues collected.

To examine the presence of expressing cells Fc? RI in chronic skin inflammation in humans, biopsies of chronically inflamed skin from patients with atopic dermatitis (AD) were collected. The di-agnostic of AD was made according to the criteria of Hanifin and Raijka (Hanifin, J. M. and Rajka, G (1980) Acta Derm. Venerol.

(Stockholm) 92: 44-47). The atopic patch test (APT) was performed as described in Langeveld-Wildschut, E.

G., et al., (1995) J. Allergy Clin. Immunol. 96: 66-73. In summary, a strip of the skin and the allergen Dermatophagoides pteronyssinus (Haarlem's) was detached 10 times.

Allergenen Laboratorium, Haarlem, The Netherlands; 80? L, ,000 AU / ml) was applied using Leucotest (Beiersdorf, Hamburg, Germany) on clinically normal skin of the back of patients diagnosed with AD. In analogous skin, sodium lauryl sulfate (SLS, Sigma, 0.1% in saline) was applied in a similar way. A mild polyphologic eruption (PLE) was diagnosed based on the polymorphic clinical image, with presence of papules and vesicles, severe pruritus and clinical response after WA and / or WB irradiation.

The unexposed skin was irradiated with six doses for minimal erythema using a Philips TL12 UVB source. Sections of human skin were stained immunohistochemically using FcγRI antibodies. Cells expressing FcγRI were detected resulting from staining pink / red and against staining with hematoxylin. In normal, unaffected skin, some cells expressed Fc? I. These cells were located mainly in the dermis.

On the contrary, abundant expression of FcγRI in dermis was observed in chronically injured skin, for example skin with atopic dermatitis. The stained cells were located in infiltrates and dispersed through the dermis. No significant staining of the epidermis was observed. Next, biopsies were collected from the acute phase models, such as 24 hours after the atopic patch test (APT)., 72 hours after mild polymorphic skin rash (PLE) and 48 hours after treatment with sodium sulfate lauryl (SLS). These biopsies produced similar results, however, the number of cells expressing Fc? RI was somewhat higher than in chronically affected tissues. The presence of large numbers of cells expressing Fc? RI in acute and chronic phase is indicative of a function for these cells in the cutaneous inflammatory response.

EXAMPLE V: ESTABLISHMENT OF A MURINE MODEL FOR CHRONIC CUTANEOUS INFLAMMATION To determine whether the elimination of inflammatory macrophages from the skin is feasible and has a beneficial effect on skin inflammation, H22-R was tested in experimental animals. The induction of chronic skin inflammation was studied using shaved skin from transgenic hFc? RI mice and their non-transgenic baits after repeated topical application of SLS. The expression pattern, gene regulation and function of hFcγRI in these mice mimic that of humans (Heijnen, I.A., et al., (1996) J. Clin.Invest.97: 331-338). Some protocols were tested and the daily application of 5% SLS for 10 days was tested appropriately as described in the section entitled Materials and methods. Low numbers of T cells, dendritic cells and macrophages were detected in normal, untreated skin (5 + 4, 7 ± 4 and 15 ± 3 per mm, respectively). In addition, some cells expressing hFc? RI were detected in normal, untreated skin (5 ± 2 per mm2), and the distribution was similar to that of normal, unaffected human skin. Treatment with SLS gave rise to the thickening of the epidermis and a vast dermal infiltrate consisting of T cells, dendritic cells and macrophages (Figure 3A). For these experiments a single intradermal injection of IT was administered to chronically inflamed skin and at different intervals, biopsies were taken by function and stained immunohistochemically. The number of cells expressing hFc? RI also increased drastically (Figure 3A) (75 + 11 per mm2) and equal in chronically affected human skin, these cells were distributed mainly in the dermis. There was no significant difference in cellular composition between hFc? RI and non-transgenic transgenic mice. However, no significant cell staining for hFc? RI was observed in the latter. No detectable presence of cells expressing hFc? RI or macrophages was observed after injection alone with H22-R. The similarities with respect to the cellular composition and the expression of hFc? RI between chronically inflamed human skin and SLS-induced inflammation in hFc? RI transgenic mice makes this model convenient to study the function of cells expressing hFc? RI during chronic skin inflammation. This model, in combination with the H22-R IT was used in the examples set forth below. EXAMPLE VI: EFFECTIVE DEPLETION OF MACROPHAGES EXPRESSING F? RI IN VIVO To determine if H22-R was as effective in killing cells expressing live hFc? RI m as was demonstrated by HIV, H22-R was injected mdermically in mice treated with SLS. Chronic skin inflammation was induced in transgenic mice expressing human FcγRI by repeated topical application of an irritant, 5% sodium lauryl sulfate as described in the section entitled Materials and Methods, supra. Two injections μl intradermal, 2 × 10"M (3 μg of H22 and 0.6 μg of Ricin A) were administered once to the SLS-treated skin of transgenic hFc? RI and non-transgenic mice. laterally, 5 identical vehicle control injections were administered, SLS was continued while at different time points skin samples were collected, drained of lymph nodes, liver and spleen for immunohistochemical analysis.

The localized nature of the intradermal injections was examined by detecting the uptake of the carbon particle by the macrophages. A cut of the murine skin after intradermal injection of the carbon particles revealed the presence of carbon particles mainly in the dermis, but not under the musculature cutaneous. This distribution demonstrates the localized nature of the intradermal injections. A representative immunohistochemical cut of the skin of transgenic mice expressing human FcγRI after repeated topical applications of sodium lauryl sulfate, and intradermal injection with vehicle control or Ricin A-H22 revealed the thickening of the epidermis and a large number of infiltrating cells in the dermis, 24 hours after treatment. This staining pattern indicates chronic inflammation induced by the irritant. For the most part, infiltrating cells detected were positive macrophages for Fc? RI (dyed in pink). In contrast, staining infiltrated cells that expressed the Fc receptor? was significantly reduced 24 hours after the injection of the Ricin A-H22 immunotoxin. The disappearance of cells expressing hFcγRI from the skin was detected within 24 hours of IT exposure (Figure 3A). In spite of the continuous application of SLS, the depletion was still complete approximately 96 hours, after which repopulation occurred. The repopulation was complete only until 120 hours (Figure 3A). In the drainage of the lymph nodes, liver and spleen, no significant changes in the expression of hFc? RI were observed. This observation highlights the fact that the effect remains limited to the injection site. In the site injected with the vehicle control, no significant changes were observed in the non-transgenic mice. The rapid and almost complete disappearance of cells expressing hFc? RI and their absence from the skin showed the practicability of H22-R IT to eliminate cells expressing hFc? RI in chronic cutaneous infiltration in vivo.

EXAMPLE VII: EFFECT OF DEPLETION OF CELLS EXPRESSING HFRIRI IN LOCAL CUTANEOUS INFLAMMATION At the same time with the reduction of cells expressing hFc? RI, the abundance of the macrophages that express MOMA-2. This finding demonstrates that the injection of H22-R causes efficient depletion of inflammatory macrophages of the affected skin (Figure 3A). In contrast, no significant change in the populations of the macrophages occurred in non-transgenic mice. This selective depletion confirms the specificity of H22-R in the orientation and elimination of the macrophages of the skin.

To further assess the localized nature of the depletion of macrophages, hematopoietic tissues such as lymph nodes, spleen and liver were examined. No important cell depletion by the immunotoxin was observed in other hematopoietic tissues. The identical treatment of non-transgenic baits gave rise to undetectable changes in any of the populations of cells examined. These results indicate that the depletion of the macrophages was specific for human Fc? RI carrier cells and remained limited to the injection site. The specificity of the procedure for the elimination of macrophages locally is further demonstrated by the disappearance, during the first 24 hours, of the macrophages, while no significant depletion was observed in dendritic cells, T cell populations or Langerhans cells during the points of time examined. Injections of H22-R had no direct effect on the number of T cells and dendritic cells (Figure 3B). However, after the disappearance of the macrophages expressing hFc? RI, the numbers of T cells and dendritic cells began to decrease in the skin. The reduction of numbers in T cells and dendritic cells indicates the resolution of local inflammation and thus a beneficial effect of the deletion of inflammatory macrophages in local inflammation, even in the presence of continuous inflammatory stimulus (Figure 3B). These findings demonstrate the efficacy and specificity of CD64 IT in the depletion of inflammatory macrophages of the skin at the histological level. The subsequent disappearance of other inflammatory cells indicates a deleterious function of macrophages in chronic cutaneous inflammation.

EXAMPLE VIII: LOCAL DEPLETION OF MACROPHAGES ORIGINATES CLINICAL IMPROVEMENT OF THE SKIN To determine if the local depletion of the macrophages causes clinical improvement of the skin, two parameters were measured: local temperature of the skin erythema. The erythema is mainly due to the increase in capillary dilation, and is directly related to the increase in skin temperature. To detect changes in skin temperature induced by SLS application and IT injection, the Animals were immobilized by moderate sedation with ether and the local temperature was measured using a skin probe? (Ellab A-HI, Denmark). To discover capillary dilation and vascular leakage as a parameter for inflammation, the animals were sedated with ether and injected intravenously with 1% Evans blue solution. After 15 minutes, the animals were sacrificed and the skin removed for evaluation. By using a small probe for skin, the I 10 local changes in skin temperature were measured in animals treated with IT and control animals. An increase in temperature after treatment with SLS was detected confirming the induction of local inflammation. Figure 4A is a bar graph depicting the effect of the thermometric injection of H22-R on the local temperature of the skin as a function of time. A fall in temperature reaching levels comparable with untreated, unaffected skin [sic]. This decrease in temperature in animals treated with IT was commonly detected lasting 96 hours. After this time, the temperature increased again, reaching levels comparable to those presented before the IT injection. These changes in temperature are indicative of the resolution of inflammation. Neither the vehicle controls nor the non-transgenic mice showed a similar decrease in the temperature. In addition, a close temporal correlation was observed between the disappearance of the macrophages and the decrease in the local temperature of the skin. On the contrary, with the reappearance of the macrophages an increase in temperature was detected. These findings strongly suggest a crucial function of macrophages in local inflammation. In mice, the skin growth is difficult to assess due to the thinness of the murine skin. To facilitate the visualization of local capillary dilation, Evans blue is injected intravenously to these animals. For these experiments, chronic cutaneous inflammation was induced in transgenic mice hFc? RI (n = 9) or non-transgenic mice (n = 9) by epicutaneous application of SLS and IT or vehicle control administered via mderdermica. Evans blue was injected intravenously at 24 hours and 30 minutes after the animals were sacrificed and the skin of the midsection was separated. Using this technique, the presence of an inflammatory response was detected after treatment with SLS. No significant effect of IT on capillary dilation was detected in non-transgenic mice or vehicle control. On the side injected with H22-R of the latter, however, the injection site itself was not painted blue showing resolution of -OR-. local inflammation. In addition, the total intensity of the blue stain was lower on the side injected with H22-R.

EXAMPLE IX: PROLONGED SUPPRESSION OF IN VIVO INFLAMMATION WITH REPEATED INJECTIONS OF CD64-IT To establish if IT could be used for a long time, the skin temperature was measured daily and with the increase, the animals were again injected into the skin. same place. During the experiment the application of SLS was continued. Figure 4B shows that the inflammation can be controlled for at least 18 days in transgenic hFc? RI mice injected only with H22-R. Control with vehicle and non-transgenic mice did not show a significant decrease in temperature at any point in time reviewed. Repeated injections with IT showed that it was possible to suppress inflammation for a long time. These findings demonstrate the applicability of prolonged IT treatment in patients with chronic cutaneous inflammation. Taken together, these experiments show a beneficial effect of the local elimination of the macrophages in chronic cutaneous inflammation induced by the application of SLS. In summary, the experiments described in the examples herein show that activated macrophages can be eliminated selectively and eliminated in a effective using the methods and compositions of the present invention without significantly affecting other cutaneous or hematopoietic cell populations. In addition, the effects of the immunotoxin remain primarily located in the delivery area, thus reducing the negative systemic effect in other cells expressing FcγR. The reduction in inflammation with the elimination of macrophages highlights the importance of inflammatory macrophages as an agent in the induction and maintenance of skin inflammation. A reduction in inflammation is detected histologically, as well as by decreases in clinical parameters such as local temperature of the skin and redness of the skin. In addition, repeated application causes suppression of inflammation for prolonged periods. The prolonged efficacy suggests the potential use of the methods and composition of the present invention in the management of local cutaneous inflammation in patients suffering from chronic skin diseases. This method described in the present may have wider applications since inflammatory macrophages probably play an important role in the chronicity of other types of chronic inflammation, such as rheumatoid arthritis.

Staining for CD64 in human skin showed numerous cells expressing FcγRI during acute and chronic cutaneous inflammation. This observation indicates that the orientation of the macrophages through Fc? RI can actually provide a new therapeutic approach for cutaneous inflammatory disease in humans. In fact, the effective reduction in chronic inflammation induced by SLS in Fc? RI transgenic mice shown herein supports the potential therapeutic uses of these immunotoxins.

Equivalents Those skilled in the art will be aware, or will be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are proposed to be comprised by the following clauses.

Claims (33)

1. A method for selectively reducing the number or activity of macrophages consists in contacting macrophages with a compound that binds to macrophages consisting of: (a) an agent that binds to an Fc receptor, where Fc receptor binding is not blocked by endogenous antibodies; and (b) an agent that kills or reduces the activity of macrophages.

2. A method of treating or preventing a disease in an individual, characterized by aberrant activity or number of macrophages within a selected area of the individual, consists of locally administering in the area a compound that binds to macrophages consisting of: (a) an agent that binds to an Fc receptor, wherein the binding to the Fc receptor is not blocked by endogenous antibodies; and (b) an agent that kills or reduces the activity of macrophages.

3. The method of claims 1 6 2, wherein the agent that binds to the Fc receptor binds at a site that is not bound by an endogenous IgG or IgA.

4. The method of any of claims 1 or 2, wherein the Fc receptor is an Fc receptor? (Fc? R) or an Fc receiver? (Fc? R).

5. The method of claim 4, wherein the Fc? it is selected from the group consisting of Fc? RI, Fc? RII and Fc? RIII.

6. The method of claim 5, wherein the Fc? It is a human Fc? RI.

The method of claim 4, wherein the Fc receptor is a human Fc? R receptor.

The method of any of claims 1 or 2, wherein the compound that binds to the macrophages consists of an anti-Fc receptor antibody conjugated to a toxin.

9. The method of claim 8, wherein the anti-Fc receptor antibody is an anti-Fc receptor? or a fragment of it.

10. The method of claim 9, wherein the anti-Fc? Receptor antibody. is a monoclonal antibody selected from the group consisting of mab22, 32 and 197, or a fragment thereof.

11. The method of claim 9, wherein the anti-Fc? Receptor antibody. is a humanized IJ22 antibody produced by the cell line having accession number ATCC CRL 1117 or a fragment thereof.

The method of claim 8, wherein the toxin is selected from the group consisting of Gelonin, Saporin, Exotoxin A, Onconase and Ricin A.

13. The method of claim 1, wherein the agent that kills or reduces the activity of macrophages is encapsulated within a liposome.

The method of claim 13, wherein the agent that kills or reduces the activity of a macrophage is dichloromethylene diphosphonate (CL2MDP) or derivatives thereof.

15. The method of claim 13, wherein the agent that binds to an Fc receptor is a single-chain antibody.

16. The method of claim 13, wherein the agent that binds to an Fc receptor is an anti-Fc receptor antibody. or a fragment of it.

17. The method of claim 13, wherein the agent that binds to an Fc receptor is an anti-Fc receptor antibody. single-string or a fragment thereof.

18. The method of claim 1, wherein the step of contacting occurs in the culture.

The method of any of claims 1 or 2, wherein the compound that binds the jpacrofagos is administered topically, intradermally or subcutaneously in a pharmaceutically acceptable carrier.

The method of claim 2, wherein the disease is characterized by enhanced proliferation and / or secretion of macrophage growth factor.

21. The method of claim 2, wherein the disease is selected from the group consisting of: psoriasis, atopic dermatitis, scleroderma, cutaneous lupus erythematosus, human immunodeficiency virus infection, multiple sclerosis, rheumatoid arthritis, chronic mild polymorphic dermatosis, pulmonary diseases Chronic obstructives and granulomatoci of Wegener.

22. A method for diagnosing a disease in an individual, characterized by numbers or aberrant activity of the macrophages, consists of: contacting a biological sample of the individual with a compound that binds to the macrophages consisting of an agent that binds to an Fc receptor, where the binding to the Fc receptor is not blocked by endogenous antibody; and detecting the level of binding to the Fc receptor as an indication of the amount of the Fc receptor protein in the sample, wherein the elevated expression of the Fc receptor protein, or an increase in the number of macrophages expressing the protein of the Fc receptor is indicative of a disease mediated by macrophages.

23. The method of claim 22, wherein the compound that binds to the macrophages also contains a detectable label.

24. The method of claim 22, wherein the expression of the Fc receptor protein is detected by autoradiographic, colorimetric, luminescent or fluorescent detection.

The method of claim 22, wherein the disease is selected from the group consisting of psoriasis, atopic dermatitis, multiple sclerosis, scleroderma, cutaneous lupus erythematosus, human immunodeficiency virus infection, chronic mild polymorphic dermatosis, chronic obstructive pulmonary diseases and Wegener's granulomatocis.

26. A method for selectively reducing the number or activity of macrophages consists in contacting macrophages with a compound that binds to macrophages and consists of: (a) an agent that binds to a receptor Fc; and (b) an agent that kills or reduces the activity of macrophages, wherein the agent that kills or reduces the activity of macrophages is encapsulated within a liposome.

27. The method of claim 26, wherein the agent that kills or reduces the activity of macrophages is dichloromethylene diphosphonate (CL2MDP) or derivatives thereof.

28. The method of claim 26, wherein the agent that binds to an Fc receptor is a single-chain antibody.

29. The method of claim 26, wherein the agent that binds to an Fc receptor is an anti-Fc receptor? or a fragment of it.

30. The method of claim 26, wherein the agent that binds to an Fc receptor is an anti-Fc receptor antibody. single-string or a fragment thereof.

31. One method to selectively reduce the number or activity of macrophages is to contact macrophages with a compound that binds to macrophages and consists of: (a) an agent that binds to an Fc receptor; and (b) an agent that kills or reduces the activity of the macrophages, wherein the compound that binds to the macrophages is administered topically, subdermally or subcutaneously in a pharmaceutically acceptable carrier.

32. A method for treating or preventing a disease in an individual, characterized by aberrant activity or number of macrophages within a selected area, the individual, is to locally administer to the area a compound that binds to macrophages and that consists of : (a) an agent that binds to an Fc receptor; and (b) an agent that kills or reduces the activity of macrophages, wherein the compound that binds to the macrophages is administered topically, intradermal or subcutaneous in a pharmaceutically acceptable carrier.

33. The method of claim 32, wherein the disease is characterized by enhanced proliferative growth and / or secretion of the macrophage.