JPH06505253A - Monoclonal antibodies against ELAM-1 and their uses - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Monoclonal antibodies against ELAM-1 and their use Background of the invention The present invention is for the treatment of inflammation and other pathological conditions mediated by intercellular adhesions. The present invention relates to compositions and methods. In particular, the present invention focuses on cell surfaces involved in intercellular adhesion. Novel immunity that selectively binds to a functional epitope on the receptor (ELAM-1) This invention relates to inhibiting cell adhesion by using globulin.

Vascular endothelium is a vascular endothelial cell that contains specific cells prior to their migration across the vessel wall and into the surrounding tissue. It plays a central role in the adhesion of certain cells in the blood stream. for example , certain pro-inflammatory substances, such as bacterial endotoxins, tumor necrosis factor, and - Leukine l acts directly or indirectly on the vascular endothelium to promote leukocytes and lymphocytes. Promotes adhesion of the ball. These cells then pass through the blood vessel wall and become damaged. or move to the infected area. Adhesion of cells to the inner wall of blood vessels is associated with tumor metastasis. can also be considered. Circulating cancer cells probably take advantage of the body's normal inflammatory mechanisms. It then binds to areas of the blood vessel wall where the endothelium is activated.

Latest research shows that specialized cell surfaces on endothelial cells, platelets, and white blood cells receptors (also known as LEC-CAMs, LECAMs or 5electins) It is clear that the It has become. These receptors have a lectin-like domain, epidermal growth factor Surface glycoproteins with sites of homology and sites of homology to complement regulatory proteins It is white (see Incorporated by Bevilacqu). For example, LECAM-2 A selectin called ELAM-1 is the first step in many inflammatory responses. has been shown to mediate the adhesion of endothelial leukocytes to activated endothelial cells. It's turning into a crab.

In particular, ELAM-1 is derived from the human neutrophil, monocyte, and promyelocyte cell line HL-60. It has been shown that it binds to

This common class of cell surface receptors can be expressed on a variety of cells. Wear. For example, GMP-140 (PADGEM, LECAM-3 and CD62 ) are present on the surface of activated platelets, where platelets is another selectin receptor that mediates the interaction between leukocytes and leukocytes. Similarly, L.A. M-1 (also known as LECAM-1) is a circulating lymphocyte, It is a constitutively expressed cell surface receptor and a “homing” receptor in lymph nodes. Work as a puta.

In order to interfere with the action of these receptors and therefore inhibit cell adhesion, Various methods have been developed. For example, monochrona directed to ELAM-1 Antibodies have been proposed as adhesion inhibitors between endothelium and leukocytes. however However, the relationship between inhibition of adhesion by antibodies and in vivo efficacy is not clear. did Therefore, the use of antibodies reactive with ELAIJ-1 could be used to effectively treat inflammatory diseases. There is a demand for evidence of medical treatment.

Summary of the invention The present invention provides a method for binding to a functional epitope on ELAM-1 and thereby The present invention provides an immunoglobulin that has the ability to suppress cell-cell adhesion.

In particular, this immunoglobulin is involved in various inflammatory disease responses mediated by ELAM-1. For example, treatment of septic shock, respiratory distress syndrome in adults or injury-related sepsis It is effective in A particularly preferred immunoglobulin is A, which was deposited under the Budapest Treaty on the 1st.

Secreted by a cell line designated T, C, C, accession number HB10591. It will be done. The present invention provides pharmaceutical compositions for treating or diagnosing inflammatory reactions in patients. provide a method. This composition is preferably administered intravenously. effective treatment The dosage is preferably from about 1 mg/kg body weight to about 20 mg/kg body weight. Between about 5 mg/kg body weight and about 15 mg/kg body weight. This composition is It can also comprise targeted liposomes embedded with immunoglobulins. this The liposome can comprise an anti-inflammatory chemotherapeutic agent. This immunoglobulin A drug is typically labeled when used as a diagnostic agent.

Brief description of the drawing Figure 1 shows that prophylactically administered monoclonal antibodies against ELAM-1 This study shows that the present invention prevents ribopolysaccharide-induced lethality in humans.

Figure 2 shows that therapeutically administered monoclonal antibodies against ELAM-1 This study shows that the present invention prevents ribopolysaccharide-induced lethality in humans.

Figure 3 shows monocloners of the present invention that suppress boteichoic acid-induced pleurisy in rats. This example illustrates the capabilities of the antibody.

Figure 4 shows the ribopolysaccharides and peptides required to induce neutrophil adhesion to human endothelial cells. The optimum concentration of ribotic acid and ribotic acid is shown.

Figure 5 shows the time course of ribopolysaccharide and ribotic acid-induced neutrophil adhesion and this Figure 3 shows that adhesions are inhibited by antibodies against ELAM-1.

Figure 6 represents the blood purification curve of the monoclonal antibody of the invention, EB3-1.

Figure 7. EB3-1 and modifications in ribopolysaccharide-induced death in rats. To illustrate the effect of form.

Description of preferred embodiments The present invention provides compositions and methods for suppressing inflammation and other diseases associated with ELAM-1. and methods. In particular, the present invention provides that cells in vivo are Utilizes immunoglobulins that have the ability to inhibit mediated adhesions. Immunization of the present invention Globulin selectively binds to functional epitopes on ELAM-1 and Interferes with leukocyte adhesion to the ductal endothelium. The present invention provides the preparation of this immunoglobulin, and The same immunoglobulin that specifically suppresses intercellular adhesion mediated by ELAM-1 and ELAM-1 Also provided are methods for screening assays to determine the identity of the patient. In addition, these things Quality diagnostic and therapeutic uses are provided.

As mentioned earlier, selectins are unique glycoproteins expressed on the surface of various cells. It is white. For example, endothelial leukocyte adhesion molecule 1 (ELAM-■) is inducibly expressed on cells (Bevilacqua et al., Kaiqi and He5sio n et al., Proc, Nat'1. Acad Sci, 87:1673-16 77 (1990), both incorporated herein by reference). This receipt inflammatory cytokines such as interleukin lβ (L!β) and tumor inflammatory cytokines. Incorporation into death factor alpha (TNFα) as well as bacterial endotoxins (ribopolysaccharides) ). These substances act directly on endothelial cells in vitro and induce polymorphonuclear of white blood cells (granulocytes), and monocyte adhesion (Bevi 1ac qua et al., Proc, Natl, Acad, Sci, RE).

Latest studies provide evidence of oligopolysaccharide ligands recognized by ELAM-1. Ta. This ligand is known as sialic acid Lewis X (SLX). , a terminal structure found in glycoproteins and glycolipids on the surface of granulocytes. See below, Ph. 1llips et al., 5science, 250:1130-1132 (1990 ); and as mentioned above, GMP-140 inhibits platelet and endothelial secretory granules. It is a membrane glycoprotein (Geng et al., Nature, 343 Nia 57-760 ( 1990), which is incorporated herein by reference). GMP-14 on the surface Activated platelets expressing 0 are transferred to monocytes and neutrophils (Jungi et al., Blo. od 67:629-636 (1986)), and further monocytoid cell lines, e.g. For example, HL60 and U937 (Jungi et al. , J. Cl1n, Invest, 79:867-874 (1987)). are known to bind, and are incorporated herein by reference in their entirety. Ru.

GMP-140 is an alpha granulosa protein with a molecular weight of 140.000, and is an alpha granulosa protein with a molecular weight of 140.000. Upon stimulation and granule secretion, expressed on the surface of activated platelets (Hsu Lin et al., J. Biol. Chem., 259:9121-9126 (198 4); Stenberg et al., J, Ce1l Biol, H:880-8 86 (1985); Berman et al., J, Cl1n, [vest.

a (1987)), Weibel-Palade in vivo (Bonfanti et al., B 73:1109-1112 (1989)).

U.S. Pat. No. 4,783,330 to Furie et al. A monoclonal antibody is described. All of the following are cited as references Incorporate it into the detailed description.

The third securen receptor is the lymphocyte homing receptor (LHR, gp”MEL ( mouse) or LAM-1 (human)).

See below, Ce1l Biology, 1:913-919 (1989), and La5ky et al., Ce1l, 56, 1045-1055 (1989). All incorporated herein by reference. In addition to lymphocyte homing, LHR It is believed to function earlier in the disease response and mediate the binding of neutrophils to the endothelium. ing. The structure and function of secretin receptors are similar to each of the above receptors. This has been revealed by cloning and expression of the full-length cDNA encoding the code (Higashio). , for example, Bevilacqua et al., 5science, HE ([ELAM -1), Geng et al., LE (GMP-140), and La5ky et al., fFE , (MEL-14)), the extracellular part of selectin is linked to the proteins described above. It can be divided into three segments based on homology. N-terminal region (approximately 20 a amino acids), mammalian C-type receptors, including the low affinity IgE receptor CD23. Related to the cutin protein family. Residues 121-155 of epidermal growth factor (EGF) Associated with proteins containing motifs. After the domain of EGF becomes three-sixths, it Repeated tandem motifs of approximately 60 amino acids each are found in the family of complement regulatory proteins. It has to do with what happens.

The immunoglobulin of the present invention recognizes and aligns functional epitopes on ELAM-1. selectively binds to and thereby promotes intercellular healing in in vitro assays. suppress wear. The exemplary immunoglobulins described herein are within the scope of the invention. in various standard screening procedures to identify additional immunoglobulins. can be used. Furthermore, the in vivo evidence provided herein is , the claimed antibodies are also effective in treating inflammatory conditions mediated by ELAM-1. prove that. As used herein, "functional epitope" refers to This is interpreted as the antigenic site on ELAM-Rusebuta bound to. Here, the antibody is , substantially inhibits the binding of ELAM-1 ligand to its ELAM-receptor. control and thereby suppress the inflammatory disease response in the patient. Eye of this specification Because of the objective, "substantial inhibition" means at least about 60% inhibition, preferably about 7% inhibition. 0% to about 90%, and more commonly about 99% or more (see below) (as determined in an in vitro assay as described).

Numerous antibodies have been identified by screening activated endothelial cells . See below, Henjin 4, Bevi 1acqua et al., Proc, Nat' 1. Acad, Sci, supra; Pober et al., J. [mmunol, 136:1680 (1986); Graber et al., 8 (1990): and PC T publication number W090105786. WO90105539 and WO90/133 00, all of which are incorporated herein by reference.

The claimed immunoglobulins are those of ordinary skill in the art for the production and manipulation of various immunoglobulins. suitable for modification, using many techniques available to the individual. How many possible modifications is discussed in more detail below.

The basic immunoglobulin structural unit is known to consist of a tetramer. There is. Each tetramer is composed of two pairs of polypeptide chains, each with The pair is one "light" (approximately 25kD) and one "heavy" M (approximately 50-70kD). D). The N-terminus of each chain reacts first to recognize the antigen Diverse sites of about 100 to lIO or more amino acids that can be Define. The C-terminus of each chain is the first to react due to the effector mechanism. Define certain parts that can be

As used herein, the term "immunoglobulin" refers to immunoglobulin genes consisting of one or more polypeptides substantially encoded by It is interpreted as a protein that is Recognized immunoglobulin genes include kappa, lambda, and a The genes of certain parts of rupha, gamma, delta, nibsilon, and mu are arranged in contains genes for various parts of its myriad immunoglobulins. The L chain is kappa or is classified as a lambda. H chains are gamma, mu, alpha, delta, and is classified as nibsilone, which, in turn, is a class of immunoglobulins. However, [gG, IgM, [gA, [gD and IgB] are defined, respectively. to justify

In addition to all antibodies, immunoglobulins include, for example, Fv, Fab, and F(a b') can exist in various forms including z as well as in single chains (e.g. , Huston et al., Proc, Nat, Acad, Sci, U, S, A, 8 5:5879-5883 (1988) and Bird et al., 5science, 24 2:423-426 (1988), and Hunkapil Her and Ho od, Nature, 323:15-16 (1986), all of these (incorporated herein by reference).

Treatment with the protease papain splits the molecule into three fragments. , two of which are named Fab fragment and the other as Fc fragment. I can't stand it. The Fab fragments contain the antigen-binding domain and C81 domain, respectively. Consists of main parts. In addition, proteolytic digestion of Fab fragments from the variable region Frees Fv fragments that are only configured. Pepsin, a protease, By cleaving H#I near amino acid residues 234 and 333, F(ab')2 and pFc'Fc fragment. F1 composed of C,2 and C,3 domains Fragments are parts of immunoglobulin molecules that mediate various effector mechanisms It's a minute. Various effector mechanisms exist depending on the H chain of this immunoglobulin. Exists. These precede complement fixation, B cell stimulation, circulatory lifespan, and phagocytosis. Involves binding of Fe receptors on standing granulocytes and macrophages. See below, generally F. undementsl is incorporated herein by reference.

The immunoglobulins of the invention can be made in a variety of ways. non-human monochrome Nal antibodies, such as murine, rabbit, horse, etc., are well known and For example, isolated ELAM-Rusebuta, activated endothelial cells, or E. The above animals containing cells transformed with DNA encoding LAM-1 were immunized. It can be completed by becoming an epidemic. obtained from the immunized animal The antibody-producing cells obtained can be immortalized and screened or used to produce the desired antibody. first screened for and then immortalized.

For a discussion of general procedures for monoclonal antibody production, see Harlow and Lane, Antibodies, A Laboratory Manual (1988), which are incorporated herein by reference.

For suitable methods for making antibodies against ELAM-1, see below, e.g. , Bev acqua et al., Proc, Nat'1. Acad, Sci, Previous Note: Pober et al., J. [mmunol, 136:1680 (1986); Graber et al., J. Immunol, 145: 819-830 (1 990) ;Leeuwenberg number W090105786, WO9010 5539 and WO90/13300, all of which are incorporated herein by reference. nothing.

The monoclonal antibodies of the invention perform well in assays such as those described below in Example 1. Preferably, inhibition of adhesion mediated by ELAM-1 and other selectin receptors Screened for control ability. Ideally, this assay would be suitable for immunoglobulin enables large-scale in vitro screening of ligand-receiver A large number of Direct and indirect methods are available and known to those skilled in the art. For example, Inhibits adhesion between cancer-bearing cells, such as PMNs, and cells expressing specific selectins. The ability to do so has been determined. As mentioned above, the selectin receptor gene was cloned Therefore, this gene can be expressed in a wide variety of cells, such as CO3 cells, C) (can be inserted and expressed in S cells and the like; typically this protein St. immunoglobulins are labeled ligands that are immortalized on solid surfaces. The carrier cells and activated selectin-bearing cells are incubated together.

Inhibition of cell adhesion then detects the label bound to its surface after appropriate washing. It was measured by Among the exemplary analyzes described below, PMNs and activity activated human endothelial cells or activated platelets were used.

The immunoglobulins identified by the above analysis can be used for various pharmaceutical, diagnostic and other applications. suitable for For the treatment and diagnosis of selectin-mediated diseases described in more detail below. In addition, this immunoglobulin is compatible with other monocloners that recognize functional epitopes. can be used to screen for antibodies. Such antibodies are that the claimed immunoglobulin binds to cells expressing selectin receptors; can be identified by its ability to interfere.

However, the therapeutic efficacy of non-human immunoglobulins in humans suggests that they It has a short half-life in plasma and is often limited to eliciting an immune response in humans. determined. Therefore, it is required to modify the antibodies and improve their therapeutic utility. Will. To improve diagnosis and treatment using monoclonal antibodies, various strategies are available. See below, Waldmann, 5science 252: 1657-1662 (1991), which are incorporated herein by reference.

One method suitable for modifying the antibodies of the present invention is to modify the antigen-binding region of a non-human antibody with a human antibody. The goal is to transfer the antibody to the region of the antibody. For example, F(abo), the fragment is Can be linked to human constant regions to create chimeric antibodies. or, To create what is referred to herein as a "humanized" antibody, the hypervariable regions are human. can be connected to the framework area of Such methods are within the skill of those skilled in the art. Commonly known and, for example, U.S. Patent No. 4.816.397, U.S. Patent number 4.816.567 and European patent publications 173.494 and 239.40 0, PCT Publication No. WO/90107861 and Reichmann, L. et al., Nature, 332:323-327 (1988), All of which are incorporated herein by reference.

Chimeric and humanized antibodies typically contain immunoglobulin genes belonging to different species. is constructed by recombinant DNA technology from segments of. For example, the mouse thing The variable (V) segment of the clonal antibody gene is similar to the human constant (C) segment. for example γ1 and γ. Preferred therapeutic chimera Antibodies therefore contain V or antigen binding domains from murine antibodies as well as human It is a hybrid protein composed of C or effector domain from an antibody. Ru. However, other mammalian species can also be used.

Chimeric and humanized antibodies have many advantages over murine antibodies for use in human therapy. It has many potential benefits. For example, the human immune system uses chimeric antibodies as foreign It is not possible to recognize the C region of the body and therefore the injected Antibodies that react with mela antibodies are generally foreign mouse antibodies. There should be fewer antibodies directed. In addition, the injected mouse antibodies , much shorter than the half-life of human antibodies of the same class, almost identical to that in human circulation. would have the same half-life and be given in smaller amounts and fewer times administration.

Other methods of producing antibodies of the invention are described by Huse et al., 5science, 246: 1275-1281 (incorporated herein by reference to 1989X) Screening of DNA libraries from human B cells according to general documentation and then antibodies (or binding fragments) with the desired specificity. By cloning and amplifying the sequence encoding the human selectin receptor, Coating human monoclonal antibodies or portions thereof that specifically bind to Sebta The goal is to isolate the DNA sequence that is coded.

In one aspect, the invention provides H and/or a recombinant DNA segment encoding the variable or hypervariable region of the light chain. ing. The DNA segments encoding these regions are typically Encoding a variable region, such as a human gamma heavy chain region or a human kappa light chain region It will be connected to the DNA segment. Those skilled in the art will understand that codons are degenerate and non-essential. Amino acid substitutions cause other DNA sequences to change their DNA as described below. It will be appreciated that the A sequence can be easily substituted.

This DNA segment typically has a naturally associated or heterologous promoter. is operably linked to a chimeric antibody-encoding sequence that contains a tag site. It further includes expression control DNA sequences. Preferably, the expression control sequence is eukaryotic. in a vector capable of transforming or transfecting host cells, It would be a eukaryotic promoter system. This vector takes up a suitable host. Once introduced, the host has conditions suitable for high level expression of the nucleotide sequence. and, optionally, the L chain, the H chain, the L/H chain dimer, or will be followed by collection and purification of the complete chimeric antibody.

The native form of the "mature" immunoglobulin has one or more components within its sequence. varies somewhat in length by deletion, substitution, insertion or addition of amino acids in That is well known. Therefore, both the variable and constant regions are substantially subject to natural modifications, but still "substantially the same" and that They have been able to maintain their respective activities. Human constant region DNA sequence from a variety of human cells, but preferably immortalized B-cells or (incorporated herein by reference) according to well-known procedures. obtain. Source cells for the DNA sequence and host cells for expression and secretion. is available from numerous sources, such as the American Type Culture Collection (Ame rican Type Culture Co11ection) 5th edition (1985) Rockville, Maryla nd, U, S, A, herein incorporated by reference). can be

In addition to these naturally occurring forms of immunoglobulin chains, modifications to “substantially the same” The synthesized heavy and light chains can be prepared using various recombinant DNA techniques well known to those skilled in the art. can be easily operated and produced. For example, the chain is By methods such as amino acid substitution, terminal and intermediate additions, and deletions, may vary from its natural sequence in the sequence. Alternatively, a part of the −order structure ( usually only contains at least about 60-80%, typically 90-95%) Fragments of the polypeptide can be produced. These fragments are One or more immunoglobulin activities ( (W, tl;!', complement fixation activity). In particular, like many genes, immune Each globulin-related gene has one or more distinct biological activities. It is noted that it contains distinct functional areas with . These have novel properties. to produce a fusion protein (e.g., an immunogen) with other genes (e.g., an enzyme). ) can be fused into functional areas from In general, genetic modifications are Various well-known techniques, e.g. can be done.

The nucleic acid sequences of the invention that ultimately express the desired chimeric antibody can be synthesized from a variety of different polynucleotides. Creotides (genome or cDNA, RNA, etc.) and components (for example, ■, J, D and C sites) and by various techniques. . Combination of appropriate genomic sequences is currently the most common production method, but Additionally, cDNA sequences can also be used (see European Patent Application No. 851). 02655.8.85305604.2.84302368.0 and 8511 5311.4. and PCT application numbers GB85100392 and US86102 269. (all of which are incorporated herein by reference).

As stated earlier, a DNA sequence is a sequence that is capable of controlling expression. After concatenating into a column (i.e. placed to ensure that it works), Expressed in the host. Such expression vectors are typically episomal or It can replicate within the host's organism as an integral part of the host's chromosomal DNA. In general, expression The vector allows the detection of those cells transformed with the desired DNA sequence. specific selectable markers, e.g., tetracycline or neomycin, to (See, e.g., U.S. Pat. No. 4,704,362, incorporated herein by reference. ).

Escherichia coli (E. coli) is a particularly useful source for cloning the DNA sequences of the present invention. It is one of the nuclear cell hosts. Other microbial hosts suitable for use include Bacillus sp., e.g. For example, Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serr. atia) and various Pseudomonas species. Ru. In these prokaryotic hosts, specific expression control sequences that are compatible with the host cell (eg, an origin of replication). In addition , some of the various well-known promoters include, for example, the lactose promoter. beta-lactose promoter mechanism, tryptophan (trp) promoter mechanism, beta-lactose promoter mechanism There is a promoter mechanism, or a promoter mechanism from lambda phage. ). This promoter, optionally with an operator sequence, To typeically control expression and initiate and complete transcription and translation: 1. lipo It has the sequence of somesome binding site, etc.

Other microorganisms, such as yeast, can also be used for expression.

Saecharomyces is a preferred host and suitable Vectors may optionally contain expression control sequences, such as 3-phosphoglycerol. promoters, including protein kinases or other glycolytic enzymes, as well as origins of replication, termination Arrays, etc.).

In addition to microorganisms, mammalian tissue cell cultures may also be used to produce polypeptides of the invention. (Dongfeng, Winnaeker, “Cloning from Genes”) to” VCHPublisher, N, Y, (1987) , herein incorporated by reference). Eukaryotic cells are actually preferred, why If so, many suitable cell lines capable of secreting intact immunoglobulins are present. This is because it has been developed in the technical field, and CHO cell lines and various CO3 B-cell lines, HeLa cells, myeloma cell lines, etc., preferably transformed B-cells or or hybridoma. Expression vectors for these cells contain expression control sequences. sequences, such as origins of replication, promoters, enhancers (Queen, C, et al., 1 mmuno1. Rev., 89:49-68 (1986), cited here. (incorporated herein), as well as any necessary process information sites, such as liposome binding sites. , RNA splicing sites, polyazonylation sites, and transcription termination sequences. can be included.

A preferred expression control sequence is the immunoglobulin gene, SV40. adenovirus, This is a promoter derived from bovine papillomavirus, etc.

DNA segments of interest (e.g. heavy and short chain encoding sequences and expression control sequences) Vectors containing sequences) can be prepared using well-known methods that vary depending on the type of cell host. It can be transferred to host cells. For example, calcium chloride Transfection 3 is commonly used for prokaryotic cells. however, Calcyl phosphate Spring Harbor Press, (1989 ), which are incorporated herein by reference.

Once expressed, whole chimeric antibodies, dimers thereof, or individual antibodies of the invention and H chains by ammonium sulfate precipitation, fractional column chromatography, gel electrolysis. can be purified according to standard procedures in the art, including electrophoresis, etc. (see, Generally, 5cope, R, protein purification, Springer-Verl ag, N, Y, (1982)). -li 1, partially or as desired homogeneous Once purified, the polypeptide can then be used therapeutically or in the development of analytical procedures. 1: is used as a coloring agent for immunofluorescence tests, etc. References, Generally, Methods of Immunology, Vols, [and II, Eds, Lefk ovits and Pernis, Academie Press, New York, N. Y. (1979 and 1981)).

The composition of the present invention has 1. a selectin receptor of cell-F associated with numerous ataxias; It comprises a selectively binding monoclonal antibody. For example, many inflammatory disorders is associated with selectins expressed in defective endothelial cells. The term "inflammation" is Used in the specification to refer to responses of both specific and non-specific defense mechanisms. Ru. A specific defense mechanism response is a specific immune mechanism response to an antigen. Special Examples of different immune system responses are antibody responses to antigens, such as viruses, and delayed Including extended hypersensitivity. An example of a non-specific immune system response is a general memory of immunology. It is an inflammatory response mediated by white blood cells that cannot survive. Such cells include granulocytes and Contains macrophages. An example of a non-specific reaction is PMN leukocytes at the site of bacterial infection. (e.g., invasion of the lungs in bacterial pneumonia and pus in swelling) formation).

Inflammatory conditions that can be treated with the present invention include, for example, septic shock, injury-related sepsis, rheumatoid arthritis, post-ischemic leukocyte-mediated tissue destruction (reperfusion injury), acute leukocyte-mediated lung injury (e.g., Adult Respiratory Distress Syndrome), immune complex-mediated tissue (e.g. (e.g. lung) damage, and chronic inflammatory conditions including atopic dermatitis and psoriasis. Ru. In addition, tumor metastasis can be prevented by suppressing the adhesion of circulating cancer cells. I can do that. Examples include colon carcinoma and melanoma.

Antibodies of the invention and pharmaceutical compositions thereof can be administered parenterally, i.e., transdermally, intramuscularly. It is particularly useful for intravenous or intravenous administration. complete immunoglobe phosphorus or their binding fragments, such as Fab, F(ab')z, Others are suitable for use in pharmaceutical compositions. In addition, numerous new drug delivery applications are being developed. roaches are being developed and the pharmaceutical compositions of the present invention can be applied using these new methods. It is equally suitable for administration using See below, Langer, 5cienc. E., 249:1527-1533 (1990), herein incorporated by reference. take in.

In one embodiment, the antibodies of the invention target conventional anti-inflammatory drugs or drugs to specific sites of tissue damage. or other agents. ELAM-1 Through the use of targeted antibodies, these drugs can reach higher concentrations at the site of injury. It can be achieved. Side effects from traditional anti-inflammatory chemotherapeutics include their low dosage, damage to the by localization of the agent at the site and/or encapsulation of the agent prior to delivery. , can be substantially relaxed.

The antibody can be paired directly or indirectly with a chemotherapeutic agent. This group The combination can be performed by methods generally known in the art, and should not substantially inhibit the ability of the chemotherapeutic agent to bind to should not substantially reduce gender. Various chemotherapies are used to target can be combined with For example, anti-inflammatory agents that can be combined with Epidemic converters, platelet activating factor (PAF) antagonists, cyclooxidase inhibitors, fatty acids Contains enzyme inhibitors and leukotriene antagonists. Some preferred ingredients are , Psychosporin A, Indomethacin, Naproxen, FX-506, Mykov phenolic acid, and others. Similarly, antioxidants, such as superoxide dismut can be used to treat reperfusion injuries. Similarly, anticancer drugs such as daunomycin Targeted It is said that

Targeting selectin receptors can be amphiphilic, or in aqueous solution. It can also be achieved through bipolar molecules (polar: non-polar) that exist as an aggregate. can. Amphiphiles include non-polar lipids, polar lipids, mono- and diglycerides, sulfur Contains fatides, resolectins, phospholipids, saponins, bile acids and their salts . These molecules include emulsions and foams, micelles, insoluble monolayers, liquid crystals, and phospholipids. can exist as lamellar layers, as well as as lamellar layers. These are referred to herein as They are referred to generically as somes. In these preparations, the medical The drug is incorporated as part of a liposome along with the antibody of the invention. This lipo saw When these proteins are brought into close proximity to the affected cells, they are used for the treatment of choice. Release the composition.

The liposomes of the invention contain neutral and negatively charged phospholipids and sterols, e.g. For example, they are formed from standard vesicle-forming lipids, which generally include cholesterol. of lipids The selection may take into account, for example, the size of the liposome and its stability in the blood stream. Generally, guidance is given in consideration of the situation.

Various targeting agents (e.g., ligands, receptors and monoclonal antibodies) The targeting of liposomes used is well known in the art (see , for example, U.S. Patent Nos. 4.957.773 and 4.603.044. these (both incorporated herein by reference). Combination of targeting agent and liposome Standard methods for can be used. Liposomes targeted by antibodies can be constructed using, for example, liposomes loaded with protein A. (both incorporated herein by reference).

The charge of liposomes is an important determining factor in their clearance from blood. Yes, negatively charged liposomes have reticuloendothelial cells. extended in circulation Liposomes with a long half-life are typically preferred for therapeutic and diagnostic uses.

8. Liposomes that can be maintained in the bloodstream from 12 to 24 hours provides sustained release of anti-inflammatory agents. The liposome or antibody The half-life in plasma is also important when labeled for providing diagnostic imaging in vivo. It would be important.

Various methods are described, for example, in 5uzoka et al., Ann, Rev. Biophys. , Bioeng, 9:467 (1980), U.S. Patent No. 4,235 , 871.4, 501, 728 and 4.837.028, (these are cited as (incorporated herein) for the preparation of liposomes. It is. One method creates multilamellar vesicles of non-uniform size. This method The lipids that form the vesicles also contain suitable organic solvents to create thin lipid membranes. or dissolved in an organic system and dried under vacuum or inert gas. desired The membrane is then redissolved in a suitable solvent, such as quaternary butanol, and then to create a more homogeneous lipid mixture in powdered form that is more easily hydrated. It can be freeze-dried. This membrane is used for its targeted drugs and coated with an aqueous solution of the targeting composition (antibody) and typically It is hydrated by shaking for 15-60 minutes or more. Multilamella as result The vesicle size distribution may be altered by hydration of the lipids under stronger shaking conditions or by Smaller size by addition of resolubilizing surfactants, e.g. deoxycholic acid It is possible to shift to

The hydrating solvent is present in the internal space of the liposome in the final liposome suspension. and the targeted drug at the required concentration. Typically, drug soluble The solution contains between 10-100 mg/ml in buffered saline. . The concentration of antibody is generally between about 0.1-20 mg/ml.

Extrusion through small-pore polycarbonate membranes or asymmetric ceramic membranes is effective for reducing the size of posomes to a relatively well-defined size distribution. It is also a method. Typically, suspensions are prepared until the desired liposome size distribution is achieved. The membrane pass is repeated l or more times until the that liposo The membrane can then be extruded through a membrane with smaller pores, forming a liposome. Achieve a gradual reduction in the size of the system.

Even under the most effective encapsulation methods, suspension of liposomes at their initial size The suspension contains up to 50% or more of the drug in free (non-encapsulated) form. and a targeting agent. Therefore, the target of liposomes To maximize the benefits of targeted drugs, free drug and targeting agents must be used. is important to remove from the final injectable suspension. several ways can be used for the removal of unencapsulated material from liposome suspensions. . In one method, liposomes in suspension are pelleted by high-speed centrifugation. is removed, leaving free material and very small liposomes in the supernatant. The other way is , concentrate the suspension by ultrafiltration, and then transfer the concentration to an alternative drug-free solution. including resuspending the collapsed liposomes. Alternatively, gel filtration may be used to remove solute molecules. can be used to separate large liposome particles from

Following procedures for removal of free drug and/or targeting agent, liposomes The suspension is brought up to the required use concentration for intravenous administration. this is , liposomes can be removed by, for example, centrifugation or ultrafiltration, or drug removal. The stage of is concentrated by concentrating that suspension increasing the total volume of the suspension resuspending the liposomes in a suitable volume of injection solvent. Can be done. This suspension is then sterilized by filtration. liposome-ligand Preparations of can be administered as described below.

As previously discussed, pharmaceutical compositions (targeted liposomes or free antibodies) are particularly suitable for parenteral administration. This composition is acceptable antibodies or cocktails thereof in a carrier, preferably an aqueous carrier. It consists of Various aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.3% gel. Ricin and the like can be used. These solutions are sterile and , usually free of particulate matter. These compositions are sterilized using conventional and well-known techniques. can be done. The composition can be adapted to approximately physiological conditions, e.g. and buffers, tonicity (e.g., osmotic pressure) regulators, etc., e.g., sodium acetate, Sodium chloride, keriram chloride, calcium chloride, sodium lactate, and others. Pharmaceutically acceptable auxiliary substances can be included as required. child The concentration of antibody in these forms varies widely, i.e., usually less than about 0.5% by weight. or at least vary from about 1% to as much as 15 or 20%; First, the specific dosage selected is based on liquid volume, viscosity, etc. will be selected according to the given mode. Therefore, the typical doctor for intramuscular injections A pharmaceutical composition may be prepared containing 1 ml of sterile buffered water and 50 mg of antibody. Let's do it. A typical composition for intravenous infusion is 250 ml of sterile phosphorus. A gel solution could be prepared containing 150 mg of antibody. parenteral Actual methods of preparing compositions that can be administered to patients will be known or apparent to those skilled in the art. For example, Remington's Pharmaceuti cal 5sciences, 17th edition, Mack Publishing Company, Easton, Penn5ylvania (1985), (incorporated herein by reference).

Antibodies of the invention are lyophilized for storage and, prior to use, prepared using a suitable Can be reconstituted in a carrier. This technology uses conventional immunoglobulins, With freeze-drying and reconstitution techniques known to those skilled in the art, which can be used to It has been shown that there is.

Those skilled in the art will appreciate that lyophilization and reconstitution can lead to changes in the degree of loss of antibody activity, e.g. For example, when using normal immunoglobulin, [gM antibodies are more sensitive than IgG antibodies. ; and its use tends to lead to large activity losses. It will be appreciated that the levels may be adjusted to compensate.

Compositions containing the present antibodies or cocktails thereof may be used for prophylactic and/or therapeutic treatment. can be administered for. In therapeutic use, the composition is be administered to the patient in sufficient quantities to prevent the progression of the infection and its complications. At least partially stop it.

The amount adequate to accomplish this is defined as the "therapeutically effective dose". Ru. The effective amount for this use depends on the severity of the disease and the overall state of the patient's own immune system. will depend on the data, but a typical range is about 1. Approximately 2omg from mg/kg body weight /kg body weight, preferably from about 51T1g/kg body weight to about 15mg/kg body weight It is in. If the substances of the invention are used to treat serious disease conditions, i.e. life-threatening or potentially It is noted that it can be commonly used in life-threatening situations. It is. In such cases, it is necessary to minimize the foreign substances and to use the human chimera of the present invention. 1. Treatment in view of the possibility of rejection of "foreign substances" achieved by antibody forms Substantial overdosing of these antibodies by internists administering the drug is possible; And can be considered as preferred. .

In prophylactic use, compositions containing the present antibodies or cocktails thereof may It is administered to patients who have not yet reached a pathological state to increase their resistance. This way Such an amount is defined as a "prophylactically effective dose." In this use, accurate The amount will again depend on the patient's health status and overall level of immunity, but in general: Within the above range.

Single or multiple administrations of this composition may be selected by the treating physician. Any dosage level and pattern may be used. No matter what happens, as a medicine should provide a sufficient amount of the antibody of the invention to effectively treat a patient. . 7 The antibodies of the present invention can also be used for diagnostic purposes, e.g. to identify areas of inflammation. be able to. For diagnostic purposes, the antibody may be unlabeled or Does not have to be labeled. Unlabeled antibodies are those that are specific to the antibody, e.g. Other labeled antibodies reactive with antibodies specific for constant regions of immunoglobulins can be used in combination with a second antibody (second antibody). Or that Antibodies can be directly labeled. A wide variety of labels are available, e.g. -1 radionuclide , fluorophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands ( In particular, haptens), etc. can be used. Many types of immunoassays 1. and is well known to those skilled in the art.

For diagnostic use, compositions containing immunoglobulins or cocktails thereof are It is administered to patients who are more likely to have symptoms of inflammatory disease. Or that particular Efficacy of treatment is observed. Quantities sufficient to accomplish this are ``diagnostically effective''. Defined as "dose". .

In this use, the exact amount depends on the patient's health status and the like. There will be.

For use with antibodies of interest (this kit may also be supplied 1, so), the present invention The subject antibody composition of interest is typically delivered alone or in lyophilized form in a container. or together with additional antibodies specific for the preferred cell type. Probably. Antibiotics that can be changed or not changed into labels or toxins. The body contains buffer compounds such as tris, phosphate, carbonate, etc. in its kit. Fixatives, bactericidal agents, inert proteins such as serum albumin, and a set of instructions for use Included with. Generally, these substances are based on the amount of active antibody, approximately 5 % and then based again on the concentration of that antibody in total amount. It will normally be present as at least about 0.001%. Again, excipients The active ingredient can be present from about 1% to 99% of the total composition. It may be preferred to include diluting inert fillers or excipients. Chimera's Where a second antibody capable of binding the antibody is used in the assay, the kit will normally be present in divided vials. This second antibody is typically conjugated to the label and prepared in a similar manner using the antibody formulation described above. It will be done.

The following examples are provided by way of illustration and not limitation.

Example 1 This example shows that the monoclonal antibody of the present invention was used to detect neutrophils (P) in an intercellular adhesion assay. MNs) have the ability to prevent adhesion to activated vascular endothelial cells. shows. Human tomatozoa were obtained by parturition. 20-40cm long strips and wedges I used one without a lamp mark.

Aseptic conditions connected to a syringe. In order to remove blood and blood clots, Ca÷1M The cells were washed with g+-free phosphate buffered physiological water (CPBS). the opposite end of the tonal band is then cannulated and connected to a two-way valve, and then collagenase ( Rinse briefly with Boehringer Mannheim) 1 mg/ml, then Then, I closed the two-way valve. Next, fill the band with collagenase, remove the air, and Then, I closed the three-way valve. Place the strip in a sterile container and heat at 37°C for 15-20 minutes. Inkicoved. After incubation, the zone was mixed with 30 ml of Ca and and Mg+ in PBS, and the eluted endothelial cells were washed with additional collagen. To inhibit genease activity, add 5 ml of BGM-UV medium (C1onetic s) and 10% FCS in a sterile tube. Centrifuge the cells and BGM-UV, 10 ng/ml epidermal growth factor, log/ml hydrocol. Complete medium containing thizone, centamicin, amphotericin-B and 10% FCS resuspended in. Appropriately sized tissue culture flask (according to the length of the strip!)0 ．． 1% gelatin (endotoxin-free, isolated from bovine epidermis) for 15 minutes; Coated and excess removed at room temperature. The flask contains a suspension of endothelial cells. and placed in a 5% CO incubator. Transfer the medium to the cells It was replaced 16 hours later when it appeared to be adhering. confluent Before reaching (75%), cells were subcultured. To subculture cells, The medium was removed by vacuum inlet and the flask was freed of FCS. Rinse twice with HEPES (10mM) buffered saline, then rinse with 0.025% 2 ml of Lipsin EDTAI-2 was added to the flask and the epidermal cells were suspended. The mixture was observed under an inverted microscope until it became a cloudy liquid (30 seconds to 1 minute). Then a new complete Add medium to the flask and transfer it to a new gelatin-coated flask. It was divided 1:2 or 1:3.

To establish a monolayer of endothelial cells on a 96-well assay plate, The plates were coated with gelatin as described above. Endothelium from media passaged three times Cells were harvested from the flask using 0.025% trypsin as above. Then, it was placed at 5 x 10'/well. The cells are confluent They were allowed to multiply in order to congregate.

To induce expression of ELAM-1, the medium was poured several w at a time from the well. Remove each well and add 0.05 ml of new complete medium (in the control well). ), or fresh complete medium containing 30 ug/ml rlL-1β (Tes) w ) in ell. The plate was returned to the incubator for 4 hours.

These are called irritated endothelium.

Neutrophils (PMNs) were prepared from whole blood. 50ml of whole blood was collected from volunteers. Aspirated from the donor into a heparinized vacuum tube. 25ml of blood each This is called “Mono-Poly Resolving Medium” (Flow Labs) was layered on top of 15 ml. Place this test tube in an Rτ6000 centrifuge. Centrifuge at 2000 rpm [D] for 25 minutes at 20°C, and then The speed was increased to 2500 rpm for an additional 25 minutes. PMN layer (two floating particles The underside of the cell layer) was removed and placed into a clean 50 CC centrifuge tube. next, 20mM HEPES (Gibco) and 0.2% glucose (Fisher ) including Hanks Ba1anced 5alt Solution (E(B SS) (Gibco) was added to each tube. This tube for 3 minutes Centrifuge at 3000 rpm and HBSS/HEPES/glucose The cells were resuspended three times in buffer and counted using a hemocytometer.

To perform the assay, remove the assay plate from the incubator and , adhered cells,) IBss/HEPεS/glucose+ 5mg/ml of bovine serum albumin twice. Each monocloner to be tested 100 μl of the antibody was added to the assay plate well. Plate the antibody and incubated for 20 minutes. 5xlO' PIJN to that helmet 5 It was added as 0 μl. The plate was then incubated for 6 minutes at room temperature. Ta. Invert the plate over the sink and use a multichannel pipette to Remove non-adherent cells from the well by adding 200 μl of culture medium four times. It was removed. Remove the last wash from the wash and add solubilization buffer 5. 0 μl was added.

Citric acid buffer (24.3ml 0.1M citric acid, 10.5g1500ml +25.7ml 0.2M sodium phosphate dibase, 14.2g1500ml and SQ Hz Oto 100 m1). Contains surfactants. Incubate the plate for 10 minutes with gentle mixing. and then 50 μm of OPDA (8 mg O-phenylene-diamine , Si gma catalyst #P-1526, 8 μm of 30% H2O2 and 10 ml of SQ H20) was added to each well. Place this plate at room temperature for 1 Incubate for 5 minutes and then add 15 μl of 4N HSOa to each plate to stop the reaction. Add blank reagent to 50 μl of solubilization buffer. l, 50 μl of 0PDA solution and 4N H! So, mix and prepare 15μm. Manufactured. Remove 100 μl of the surface from each well and Transferred to Xible EL [SA assay plate (Falcon). This plate Scanned at 492 nm for 30 minutes.

Furthermore, carcinoma cells: 1 mouth 205 (COIO) (ATCC number CCL222) (known to express SLX) was used. These cells are FC receptors. cross-linking by monoclonal antibodies should not occur because stomach. This assay procedure is similar to that described above for PMNs. but However, the cells were labeled with Cr (50 μCi/cell, 30 min).

Adhesions were detected using a gamma counter according to standard procedures.

The results presented in Table 1 below demonstrate that the monoclonal antibodies of the present invention oio 205 Effectively blocking adhesion of cells to activated endothelial cells shows. Monoclonal antibody H18/7 (PCT publication number WO 90105539, supra) was incubated as a control. in the presence of antibodies The amount of binding was arbitrarily designated as a value of 1.0. The antibodies that promoted binding in this assay were Antibodies that inhibit binding have values less than 1, while antibodies that inhibit binding have values greater than 1.

EBI-5[gG, 1.42 0.08ENBI-6[gG + 1.64 0.76EBI-7 [gG, 1.48 0.10EB3-1 [gG 3 0.18 0.12EB3-2 rgG 2 0.3 0.12EBM-21 gM　O, 210, 12 EBM-41gM O, 280, 10 H18/7 1gG *a or b 1.20 0.16 Example 2 This example shows the epitope recognized by the monoclonal antibody of the present invention and the ELAM -1 (H18/7) previously identified as binding to from competitive inhibition assays used for comparison with epitopes recognized by the body. Represents data. To do this, standard procedures (see, e.g. Harlow a nd Lane, supra), H18/7 was biotinylated and HRP- Avidin staining was detected by solid phase ELISA on [L-1 activated endothelial cells.

The value given is the absorbance at 492 nm.

As shown in Table 2, the range of inhibition of 818/7 binding was determined.

Table 2 (Monoclonal antibody = Mab) Interfering Mab Biotinylated Mab H RP-avidin control 818/7 1.175 H18/7 818/7 0.309 FBI-I HI8/7 1.021 EB3-I H18/7 0.830 ENBI-6818/7 0.346 Example 3 This example shows that the monoclonal antibody of the present invention has an effect on the activity of neutrophils (PMNs) in a cell-to-cell assay. Demonstrates the ability to prevent adhesion to sexualized platelets (with GMP-140). child The results of these assays are presented in Table 3 and demonstrate the presence of these monoclonal antibodies of the invention. In the presence of PMNs or coio 205 cells relative to activated platelets Adhesions are shown. The procedure for this test is as follows: Platelets were obtained from normal human donors and treated with PGE + (100 nMX References Po1ley and Nachman, J. Exp, Med. 158:603 (1983), incorporated herein by reference). The cells were washed with plasma protein-free material. Then, without stirring at room temperature for 20 minutes, Activated with thrombin (0,250/ml; 2xlO' platelets per ml) .

For the evaluation of GMP-140-mediated adhesion to activated platelets, two Nibrate assay and liquid phase assay were used. Plate assay was previously described. Endothelial cell and neutrophil adhesion assay (DQl) Rina et al., rmmunology, 67:502 (1989), incorporated herein by reference), as amended. This is what I did. Platelet suspension (300 μm: 10”/ml) was prepared in advance. in each well of a 48-well plate coated with 0.1% gelatin. used. The plate was incubated at 37°C for 15 minutes, then at 9°C for 2 minutes. Centrifuge at 0xg and add phosphate buffered saline ( Washed twice with PBS). collected by heat to interfere with platelet FC receptors. Add 300 μl of combined [gG (20 μg/ml) to each well, The plate was then left at room temperature for 20 minutes. This plate was washed once with PBS. Wash and then plate 300 μl of the monoclonal antibody to be tested. to each well and let the broth sit at room temperature for an additional 20 minutes. I left it alone. Meanwhile, neutrophils were isolated from human whole blood by the method described above. Those , 61Cr (450 μCi added at 300111 to 3 x 10' cells, and 10% calf fetus Washed three times with RPM containing baby serum and resuspended in 2xlO'ml.

50 μl of this suspension was added to each well of the assay plate. This block The rates were centrifuged at 90xg for 2 minutes and then left at room temperature for 5 minutes.

Unbound cells were removed with three washes with PBS. and the remaining connective cells, Remove from plate with buffer containing SDS and then process for radioactivity counting. I understood.

The liquid phase assay (see [, Arsen et al., Ce1l, 63:467 (1990 ), which is incorporated herein by reference). went. 20 μl of activated platelets (2xlO″/ml) were added to placed in an rf tube. Add 20 μl of heat-aggregated [gG] and mix. After mixing, the tube was left at room temperature for 20 minutes. Add 20 μl of monoclonal antibody and the tube was left at room temperature for an additional 20 minutes. On the other hand, PMNs are Sea urchin (without radioactive label) was prepared and diluted to 2xlO@/ml. This suspension 20 μl was added to each tube and after mixing the tubes were incubated for 20 minutes at room temperature. I left it alone. Next, the adhesions are evaluated microscopically and 2 or more of platelets were recorded as the percentage of test cells bound.

Furthermore, colon carcinoma, Co1o 205 (ATCC number CCL222), (known to express ligands recognized by selectin receptors) The assay was performed using cells derived from the following. The assay procedure used is similar to that used in PMNs. It was the same as what was done.

Mah PMNs Carcinoma (Coin 205) Precipitate Purified GMP-140 Example 4 This example demonstrates the efficacy of Mab PBS-1 in an animal model of ribopolysaccharide-induced death. I will clarify. The rat strain was chosen because PBS・-1 is different from ElAM-1. Similarly, cross-reactivity was shown to occur in rats.

One day prior to use, Escherichia coli (ε, eoli) 0111:B4 (Sigma, LPS from Lot #36P4019) in a sterile, pyrogen-free saline solution. fresh from a single lot by dissolving in saline at a concentration of 5 mg/ml. Prepared. This solution was placed on ice for 30 seconds using a Tekmark sonicator. Sonicated. Immediately before use, the material was sonicated once more for 30 seconds.

Female Lewis rats weighing 200 g (+-10 g) were Ver. purchased from BreedingLabs and at least 7 days after receipt. Maintained (for adaptation). Unless otherwise noted, groups of 10 animals are used. used. All reagents were administered parenterally via the tail vein at 0.5-1.0 ml/kg. Injected. As a negative control, animals received sterile LPS-free saline; Myeloma protein in mouse 1gG3 (J306.Low pyrogen <2 ng/mggG) accepted.

The PBS-1 Dosage/Schedule Procedure is for the prophylactic administration of PBS-1 to rats. We arrived at this empirically from the pharmacokinetic data we obtained from 1. Ta.

The “minimum” LD+ao of LPS was 7.5 mg/k in these rats. It was determined that g.

In one experiment, rats were treated with iomg/kg for 1 hour before LPS treatment. Treated with P2H4. 471O of treated t=- animals were treated with LPS All 10 mice in the -0 (injected with saline) control animals died. is. During the 24-hour observation period, those that survived were LPG-treated animals. showed characteristic clinical signs.

We (1) 2-fold-elevated, and (2) the 10 mg used in the first experiment. The amount of PBS-1 in the state of ungrooved amount/kg was tested. The results are PBS- 1 is a significant effect, 80% of the animals survived the 10 mg/kg dose (Figure 1) ,showed that. The fact that an animal survived in the saline control group is due to This group of substances was as intense as in the animals in the previous experiment, 1. The challenge suggests that it is not an "attack".

Other studies have been conducted to demonstrate the therapeutic value of PBS-1. Animals! , P.S. 10 mg/kg intravenously 1 hour before or 2.4 or 6 hours after challenge. I received a message from PBS-1 on the inner porous.

Once again, one-tenth of the animals were in the saline group as well as in the J60 6 of the group treated with 1 omg/kg myeloma protein at T = -60 min (Figure 2). survived inside. However, εB3-1 was administered 2 or 4 hours after LPS. It had a significant protective effect only when treated.

conclusion Protection seen with Cytel's Mab PBS-1 (j) M-1 (- both prophylactically and therapeutically in the treatment of mediated inflammatory disease responses) Demonstrate usefulness.

Example 5 This example shows that purified PBS-1 antibody was administered to ribotic acid in the pleural cavity of rats. It shows the effect of suppressing the inflammatory response induced by (LTA). induce pleurisy The potency of LTA has been established with a linear relationship between dose concentration and neutrophil entry. . ! , TA activates the endothelium via El, AM-1, and has a positive effect in vitro. Adhesion to the mesoglobulus has also been shown (see Example 6 below).

In this example, we induced pleurisy in rats by bilateral injection of LTA into the pleural cavity. 1. In this test animal, the PBS-1 monochrome antibody was was injected into a blood vessel in the tail. After 4 hours, the cavity of 2- is irrigated and drained of the effluent. The concentration of the mesoglobules was not evaluated. .

procedure· 1.1, TA (Sigma catalyst #L-25151, ot 89F4061), 2ml in sterile, endotoxin- and pyrogen-free saline! /ml, It was then stored at -70°C. This is divided into 1.41m1 LTA and 2.43m1 raw material. A stock solution of 750 μg/ml was made by dissolution in saline. The final concentration is 30 0μg/400711/rat).

2. Ascitic fluid (Lot 6) prepared with ffl PBS-1 was 4.5+H/ml. This was then diluted with DPBS to give a final concentration of 3.33 mg #nl. per rat The dosage is 600 μI, containing 2 mg (10 mg/Mg), and 1.5 minutes 1evvis of a 93 month old female with a wave-broken t-1゜36 weight of about 195 grams. Rats were used.

All were sedated by inhalation of meth.

4. The rats were divided into three groups.

A, Control n=1. Received 200 μl of DPBS bilaterally into the pleural cavity.

600 μl of sterile, pyrogen- and endotoxin-free saline was injected into the pleura. It was received via injection into a blood vessel in the tail 1 hour after injection.

B, treated with LTA and PBS-1. n=4　200μ on both sides to the pleural cavity 150 μg of 1.TA in 1.1 DPBS was received. 600μl PBS-1 (10 mg/kg, total 2 mg) was injected into the tail vein 1 hour after injection into the pleura. was accepted through the website.

C, 20 = 4 treated with LTA only 200 μl DPB on each side to the pleural cavity 150 μg of LTA in S was received. 600 μl of DPBS was injected into the pleura. It was received via injection into a blood vessel in the tail 1 hour after injection.

5. This rat had a 30 gauge blunt tip attached to an Ice syringe. A needle was used to inject between the third and fifth ribs. Injection into the blood vessels in the tail treated by first heating under a heat lamp to make it more protruding, and It was performed using a 26 gauge needle attached to a 3 cc syringe.

6. The rat was re-caged for 3.5 hours after the injection into the tail vein (the entire time 4.5 hours).

7. The rat was subjected to inhalation of meth.

8. The peritoneal cavity was opened to expose the diaphragm. Make small incisions on both sides of the diaphragm. and use the blunt tip of an ice syringe (no needle) to add 1 ml Both sides were washed with palinated DPBS and bound.

9. The volume of each was recorded and the cells were counted and pelleted.

They were resuspended in 100 μl saline and immediately removed for differential analysis. Create a rubbed slide from each to be stained using the sex identification dye. It was.

example Examples include the bacterial products ribopolysaccharide (LPS) and riboticoic acid (LPS). TA) expresses ELAM-1 on vascular endothelial cells. Do this The ability of human neutrophils (PMNs) to bind to cultured human endothelial cells was measured following in vivo treatment with various concentrations of LPS and LTA.

Procedure = 1. LPs (Lot #36F4019) and LTA (Sigma, #L-2 515, Lot 89F4061) in saline immediately before use. Prepared by sonication at 1 mg/ml. Add this stock solution to fresh GM-UV Diluted with medium (C1onetics), 100 μg/ml 1.50 μg/n+1 ．． 25 μs/ml, 12 μg/ml, 6 μg/ml.

3μi10+t, 1.5μg/m1.0.75μg/m1.0.37μg /ml, 0.19μg/ml.

and a treatment concentration of 0.09 u g/ml. HUVEC code #code- Next, one 96-well Co5ter fresh blood was subcultured 4 times and gelatinized. The honey collected above was taken out from the incubator. Culture within each WeU The soil was removed with a Pasteur pipette and 0.2 ml of fresh GM- (C1onetics) or the same as the treatment dilution of LPS or LTA. Replaced with capacity. Each dilution was analyzed in triplicate.

2. Place the plate in a 37°C incubator with 5% COt for 4 hours. I returned it to .

3. Neutrophils (PMNs) were prepared as in Example 1. PLINs during preparation It was maintained at room temperature. 1.04×lO” of PMN was collected and added to the same buffer. resuspended in a solution containing 5 mg/ml bovine serum albumin, 6x The volume was 1O'/25 μl.

4. Remove the stimulated HUVEC assay plate from the incubator. and the wells containing 5 mg/ml bovine serum albumin (BSA). Washed twice with RPMI 1640. After the second wash, the vvell was Refill with 100 μl of the same buffer.

5. Transfer 0.025 ml of cell suspension to the stimulated assay plate. It was added to each well.

6. The plate was incubated at 37°C for 5 minutes.

7. Systematic resuspension of unbound cells using a p200 multichannel pipette, then from the wells of the assay plate by adding and removing 0.2 ml of medium. I put it out.

8. Remove all of the medium from the well and add 50 ul of solubilization buffer. liquid was added. This is a citrate buffer containing 0.1% NP-40 surfactant ( 24.3ml 0.1M citric acid, to 5g1500ml + 25.7ml 0.2M sodium phosphate dibase, 14.2g1500ml and SQ) I!　 It was composed of 100m1).

9. Incubate the plate on a rotary shaker for 10 minutes, and then 0.05 ml 0PDA solution [8 mg 0-phenylene-diamine, Sigma Catalyst #P-15261, 8 ul of 30% H102 and 10 ml of citrate buffer (Same as above) 1 was added to each well. The reaction was allowed to proceed for 15 minutes. and then 25 μl of 4N H,SO4 was added to stop the reaction. added.

10. Add reagent blank to 100 μl volume of solubilization buffer and 50 μl of 4NH2. Prepared by mixing with 0PDA solution with SOa.

11. Remove 100 μl of surface liquid from each of the 2 wells, and and transferred to flexible ELISA assay plates (Falcon). that play The samples were scanned in a spectrophotometer at 492 nm within 30 minutes.

The results of this experiment are presented in Figure 4, and show that both LPS and LTA were effective in neutrophil and human endothelial cells. It was shown to induce adhesion molecules to cells. LPS was used at 0°2 and 5 μg/ml. It was the most effective between. LTA was most effective between 5 and 50 μg/ml . At each optimal concentration, LPS stimulates 2-3 times more neutrophils than LTA. induced adhesions.

Example 7 This example shows that the neutrophils observed in Example 6 were transferred to the endothelium activated by LPS and LTA. shows that the adhesion of is mediated by ELAM-1. endotoxin using each endotoxin The optimal time for skin activation was also investigated. We investigated the activity of human neutrophils (PMNs). LPS and LPS in the presence or absence of EB3-1 at various times after addition of the oxidizing agent. The binding ability to cultured human endothelial cells activated by LTA and LTA was determined.

procedure 1.1. LPS (Lot #36F4019) and LTA (Sigma, #L -2515, Lot 89F4061) in physiological saline immediately before use. Prepared by sonication at 1 mg/ml in water. Add this stock solution to fresh GM- Diluted in UV medium (C1onet 1cs) and added lOug/ml LTA and 0.0g/ml. A treatment concentration of LPS of 2 μg/ml was made. HUVEC: 1-Following #117 Fresh blood from one 48-well Co5ter was subcultured 4 times and cultured on gelatin. The collected honey was removed from the incubator. 8 time points, T=O, At 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 18 hours and 24 hours, Remove the medium in each 6well with a Pasteur pipette, and 0.0.0 with treatment dilutions of , LPS (3 wells) or LTA (3 wells). Replaced with 5 ml of fresh GM-LIV UV medium (1onetics).

2. Place this plate in a 37°C incubator with 5% CO for 4 hours. -Reverted to .

3. Neutrophils (PIJNs) were prepared as in Example 1. PMNs were prepared during preparation. It was maintained at room temperature. They were mixed in the same buffer but with 5 mg/ml bovine serum solution. The cells were resuspended in a solution containing rubumin to 150 μl of 2×1O”.

4. Remove the stimulated HUVEC assay plate from the incubator. and the helmet was treated with 5 mg/ml bovine serum albumin (BSA). Washed twice with RPM [1640]. After the second wash, wash the well with the same Refill with 300 μl buffer. This medium was again added to each triplicate of one w on hybridoma cultures containing EB3-I Mab. Replaced with fresh liquid. Incubate the plate with antibody for 10 minutes at 37°C. , and then removed from the incubator.

5. 0.05 ml of PMN suspension was added to the stimulated assay plate. were added to each well.

6. The plate was incubated at 37°C for 5 minutes.

7. Repeat systematic resuspension of unbound cells using a transfer pipette, followed by Remove from the well of the assay plate by adding and removing 0.3 ml of Ta.

8. Remove all of the medium from the well and add 200 μl of solubilization buffer. liquid was added. This is a citrate buffer containing 0.1% NP-40 surfactant ( 24.3ml O, 1M citric acid, 10.5g1500ml + 25.7ml O ゜2M sulfur/lium phosphate 2 bases, 14.2g 1500ml and SQ [420 to 100 mI).

9. Ink cover this play 1- for 10 minutes on a rotary shaker, and then , 0.2 ml of 0PDA solution [8 mg of 0-phenylene-diamine, Sigma Catalyst #P-1526, 8 ul of 30% H20, and 10n+1 citrate buffer (I-)1 was added to each well. The reaction was allowed to proceed for 15 minutes. and then 50 l of 4N Hz SOa to stop the reaction. added to.

10. Add the reagent blank to 100 μl volume of solubilization buffer and 50 μl of 4N H ! Prepared by mixing with 0PDA solution with SO4.

If iooμl of -E supernatant was removed from each of the 2wells, and transferred it to a flexible EL [SA certified play] (Falcon). That block The River Leh was scanned within 30 minutes in a spectrophotometer at 492 nm.

The results of this experiment are shown in Figure 5, where LPS and LTA were incubated with endothelial cells. neutrophil adhesion molecules on human endothelial cells with a peak between 4 and 6 hours under Indicates to induce. This elapsed time indicates that BLAM-induced TNF and IL-1β This corresponds to that shown previously for the derivation of 1. each time tested At the intermediate point, PMN adhesions were completely inhibited by anti-ELAM-1 monoclonal antibody. Both LPS and LTA inhibited ELAM-1 on human endothelium. Indicates that expression was induced.

Example 8 This example shows that the ability of EB3-1 to suppress LPS-induced mortality is due to complement fixation. Indicates that there is no dependence on the First = -, EB3i F (ab)' 2 fragment Experiments were conducted to determine the relative interfering effects of acid-treated and acid-treated EB3-1. complete All [gG! Treatment at pH 2,560 for 60 minutes permanently destroys the ability of complement fixation. This has been shown in advance. (Winkelhake, 1゜et al., 19 80. J. Biol, Chem, 2822-2828, incorporated herein by reference. (incorporate it into the detailed document) 11(: r-labeled HL-60 cells activated by IC-1β Adhesion to endothelial cells was detected using EB3-1 monoclonal antibodies (acid-treated Kg and F(ab )’! Measured in the presence of titers of two preparations of /:. 50% less binding Control was achieved using F(ab)'2 at approximately 0.5 μg/ml. Acid treatment Approximately 2 μg/lnl of [g was required to provide equivalent adhesion inhibition.

We next radioiodinated the two modified forms of EB3-1 and then halved the The period was determined as follows.

α-phase (min) β-phase (min) Unmodified EB3-1 2 356 pH2.5 treated EB3-1 3.2408EB31 F(ah)'! 2.3 The blood purification curve of 136EB31 is shown in FIG. These data and curves Based on the upper area calculations, we determined that in the rat model of LPS-induced mortality, EB Two modified forms of 3-1 were used. We administer to the animal a lethal dose of 1, for all three forms of EB3-1 between 2 and 4 hours after PS administration. gave approximately the same peak concentration and area on the curve. Therefore, lO plat 4 glue received a lethal intravenous dose of LPS at T.0. Next, in T = 2 hours, all accepted the following form of EB3-1.

Group I - no treatment Group I [-E83-1-intravenous porous 5+ng/J at 1.0 ml/kg Group lll-Acid treatment EB3-1- Intravenous porous 5mg/kg Group I V-EB3-I P(ab)'z-7.5mg/kg T'2 hours 5.0 mg/kg T=3 hours 2.5 mg/kg T=4 hours This dosing regimen is similar to blood purification obtained with unmodified EB3-1 at 5 mg/kg. Approximate the same area under the curve. The results shown in Figure 77 indicate that the mechanism of protection is complement fixation. This suggests that there is no complicated relationship between the two. Furthermore, we demonstrated that this immune rgG3 Fixes human complement and/or mobilizes cells by binding to F0 receptors demonstrated how to abolish the potentially toxic capacity of

The present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding. However, certain changes and modifications may be practiced within the scope of the appended claims. It should be obvious.

IZ124 hours Size Tl-ELAN-1 (NG/KG) FI6. l 顛Tl-ELAM　-I FIG.2 FIG., J.

Eight GZML FIo, 4 IJ’1 process F/G, 7 international search report Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), 0A (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG. ), AT, AU, BB, BG, BR, CA, CH, C3, DE.

DK, ES, FI, GB, HU, JP, KP, KR, LK, LU , MG, MN, MW, NL, No, PL, R○, RU, SD , 5E (72) Inventor: Winkelhake, Jeffrey L.

United States, California 92106° San Diego, #323. B Zukran Strive 1220

Claims (21)

[Claims] 1. for inhibiting cell-cell adhesion mediated by ELAM-1 in patients. A method comprising: a pharmaceutically acceptable carrier and a functional epitope on ELAM-1; administering a therapeutically effective dose of a pharmaceutical composition comprising an immunoglobulin that recognizes A method comprising administering to a patient. 2. The method according to claim 1, wherein the immunoglobulin is IgG2. 3. The above immunoglobulin is A. T. C. C. Access number HB10591 2. The method of claim 1, wherein the method is secreted by a designated cell line. 4. 2. The method of claim 1, wherein said intercellular adhesions are associated with an inflammatory response. 5. 5. The method of claim 4, wherein the inflammatory response is septic shock. 6. The above inflammatory response may be associated with respiratory distress syndrome or injury-related sepsis in adults. The method described in claim 4. 7. 2. The method of claim 1, wherein said intercellular adhesions are associated with metastasis. 8. 2. The method of claim 1, wherein said pharmaceutical composition is administered intravenously. 9. The above therapeutically effective dose is about 1 mg/kg body weight to about 20 mg/kg body weight 9. The method of claim 8, wherein the method is between. 10. The above therapeutically effective dose is about 5 mg/kg body weight to about 15 mg/kg body weight. 9. The method according to claim 8, wherein the method is between 11. A method of treating a pathological response mediated by ELAM-1 in a patient, the method comprising: , a pharmaceutically acceptable carrier and an immune system that recognizes a functional epitope on ELA-1. administering to the patient a therapeutically effective dose of a pharmaceutical composition comprising epiglobulin; A method comprising: 12. 12. The method according to claim 11, wherein the immunoglobulin is IgG3. 13. The above immunoglobulin is A. T. C. C. Access number HB10591 14. The method of claim 13, wherein the method is secreted by a cell line designated as. 14. 12. The method according to claim 11, wherein the pathological reaction is an inflammatory reaction. 15. 12. The method of claim 11, wherein the pathological reaction is septic shock. 16. The above pathological reaction is respiratory distress syndrome or injury-related sepsis in adults The method according to claim 11. 17. Claim 11, wherein the immunoglobulin is embedded within a ribosome. Method described. 18. The ribosomes mentioned above may encapsulate anti-inflammatory chemotherapeutic agents. The method according to claim 17. 19. capable of binding to a functional epitope on ELAM-1 and in a patient. Immunoglobulins capable of suppressing pathological responses mediated by ELAM-1 A pharmaceutical composition comprising. 20. The above immunoglobulin is A. T. C. C. Access number HB10591 20. The method of claim 19, wherein the method is secreted by a cell line designated as . 21. 20. The method of claim 19, wherein said pathological reaction is septic shock.