SK77393A3 - Pharmaceutical mixture for treatment of illnesses by inhibition of intercellular adhesion by elam-1 - Google Patents

Abstract

Compositions and methods are provided for inhibiting inflammatory and other disease responses mediated by ELAM-1. In particular, the present invention provides immunoglobulins which selectively bind functional epitopes on ELAM-1 and block adhesion of leukocytes to activated endothelial cells both in vitro and in vivo.

Description

Technical field

The present invention relates to compositions for treatment and methods of treating inflammation and other pathological conditions mediated by intercellular adhesion. Specifically, it relates to the inhibition of cell adhesion using novel immunoglobulins that selectively bind functional epitopes of the cell surface receptor (ELAM-1) involved in intercellular adhesion.

Current state of the union

The vascular endothelium plays a key role in the binding of some cells from the bloodstream before they pass through the vascular wall into the tissues surrounding it. For example: Some inflammatory agents, such as bacterial endotoxin, tumor necrosis factor, and interleukin 1 act directly or indirectly on vascular endothelium and promote leukocyte and lymphocyte binding. These cells then move through the vascular wall and into the area of injury or infection. Cellular adhesion to vascular endothelium is also believed to be involved in tumor metastasis. The circulating tumor cells seem to utilize normal body inflammatory mechanisms and bind to those areas of the bloodstream where the endothelium is activated.

More recent work has shown that specialized surface cell receptors on edothelial cells, platelets and leukocytes (referred to as LEC-CAM, LECAM or selectins) are involved in various intercellular interactions. These receptors are surface glycoproteins with a lectin-like domain, a region homologous to epidermal growth factor, and a region homologous to regulatory regulatory proteins of complement (see: Bevilacqua et al., Science, 243: 1160 (1989), as incorporated herein by reference). For example: For a selectin called LECAM-2 or ELAM-1, it appears to mediate endothelial leukocyte adhesion to activated endothelial cells, the first step in the inflammatory response. Specifically, ELAM is found to bind human neurophiles, monocytes, and cells of the promyelocytic line HL-60.

Surface cell receptors of these generic classes can be expressed in a variety of cells. For example: GMP-140 (also known as PADGEM, LECAM-3, and CD62) is another selectin receptor that is present on the surface of activated platelets where it mediates platelet-leukocyte interactions. Similarly, LAM-1 (also known as LECAM-1) is a constitutively expressed surface cell receptor of circulating lymphocytes, and acts as a homing receptor to lymph nodes.

Various methods have been developed to block the action of these receptors and thereby inhibit cell adhesion. For example: Monclonal anti-ELAM-1 antibodies have been suggested as agents for inhibiting adhesion-endothelium-β-leukocyte. However, the relationship between antibody inhibition of adhesion and in vivo efficacy remains unclear. Thus, there is a need for evidence of effective treatment of inflammatory diseases using ELAM-1-responsive antibodies.

According to the present invention, immunoglobulins capable of binding to functional epitopes of ELAM-1 and thereby inhibiting intracellular adhesion in patients are obtained. These immunoglobulins are particularly effective in the treatment of various disease-mediated inflammatory reactions mediated by ELAM-1, such as septic shock, post-stress adult respiratory distress syndrome, or sepsis associated with injury. A particularly preferred immunoglobulin is secreted by a cell line designated A.T.C.C. Accession No. HB 10591, deposited under the Budapest Treaty on 30 October 1990.

According to the present invention, pharmaceutical compositions and methods for treating and diagnosing inflammatory reactions of a patient are provided. The compositions are preferably administered intravenously. An effective therapeutic dose is between 1 mg / kg body weight and 20 mg / kg body weight, preferably 5 mg / kg body weight to mg / kg body weight. The composition may also contain targeted liposomes into which the immunoglobulin is incorporated. Liposomes may contain anti-inflammatory chemotherapeutic agents. Typically, immunoglobulins are labeled when used as a diagnostic agent.

Fig. 1 shows prophylactically administered monoclonal antibodies against ELAM-1 to prevent lipopolysaccharide-induced rat death.

Fig. 2 shows therapeutically administered monoclonal antibodies against ELAM-1 to prevent lipopolysaccharide-induced rat death.

Fig. 3 shows the ability of the monoclonal antibodies of the invention to inhibit lipotheichoic acid-induced rat pleuritis.

Fig. 4 'shows optimal lipoplysaccharide and lipoteichoic acid concentrations required to induce neutrophil adhesion to human endothelial cells.

Fig. 5 shows the time course of neutrophil adhesion induced by lipopolysaccharides and lipoteichoic acid and that this adhesion is inhibited by antibodies to ELAM-1.

Fig. 6 provides a blood clearance profile of a monoclonal antibody of the invention, EB3-1.

Fig. 7 shows the effect of EB3-1 and its modified forms of lipopolysaccharide-induced lethality in rats.

The invention relates to compositions and methods for inhibition and other diseases to which ELAM-1 is linked. this invention uses immunoglobulins to inhibit cell adhesion in vivo mediated

This inflammatory specifically, the ability to selectins. The immunoglobulins of the invention selectively bind functional epitopes to ELAM-1 and effectively block leukocyte adhesion to vascular endothelium. The present invention also provides methods for preparing immunoglobulins and screening for identifying those immunoglobulins that specifically inhibit intercellular adhesion mediated by ELAM-1. In addition, it provides diagnostic and therapeutic uses for these agents.

As discussed above, selectins are unique glycoproteins expressed on the surface of a variety of cells. For example: the endothelial leucocyte adhesion molecule 1 (ELAM-1) is induced to be expressed (Bevilacqua et al., Supra and Hession et al., Proc. Natl. Acad. Sci., 87: 1673-1677 ( 1990) as incorporated herein by reference). This receptor has been shown to be induced by inflammatory cytokinins such as interleukin β (IL-β), alpha tumor necrosis factor (TNFalpha), and bacterial endotoxin (lipopolysaccharide) (see Bevilacqua et al., Proc. Natl. Acad. Sci. , 84: 9238-9248, as incorporated herein by reference). These substances act directly on endothelial cells to substantially increase the adhesion of polymorphonuclear leukocytes (granulocytes) and. monocyte Bevilacq.ua .. .et. al ..,. Proc. Natl. Acad. Sci. , higher).

More recent work has shown an oligosaccharide ligand recognized by ELAM-1. This ligand, known as sialyl Lewis X (SLX), is a terminal structure found on the surface glycoproteins and granulocyte glycolipids. See Philips et al, Science, 250: 1130-1132 (1990); and Walz et al., Science, 2: 50: 1132-1135 (1990).

As discussed above, GMP-140 is a membrane glycoprotein of platelets and endothelial secretory granules (Geng et al., Nature, 343: 757-760 (1990) as incorporated herein by reference). Activated platelets that express GMP-140 on their surface are known to bind to monocytes and neutrophils (Jungi et al., Blood 67: 629-636 (1986)) as well as monocyte-like cell lines, e.g. HL60 and U937 (Jungi et al., Supra; Silverstein et al., J. Clin. Invest., 79: 867-874 (1987), as incorporated herein by reference). GMP-140 is a 140,000 molecular weight alpha membrane protein expressed on the surface of activated platelets after platelet stimulation and granular secretion (Hsu-Lin et al., J. Biol. Chem., 259: 9121-9126 (1984)) Stenberg et al., J. Cell Biol., 101: 880-886 (1985); Berman et al., J. Clin. Invest., 78: 130-137 (1986)). It has also been found in magakaryocytes (Beckstead et al., Blood, 67: 285-293 (1986)) and in endothelial cells (McEver et al., Blood, 70: 355a (1987)) within Weibel-Palade bodies (Bonfanti et. al., Blood, 73: 1109-1112 (1989)). Furie et al. U. Patent No. No. 4,783,330 disclose monoclonal antibodies reactive to GMP-140. All previous references are incorporated herein by reference.

The third selectin receptor is a lymphocyte homing receptor (LHR, also referred to as gp ⁹⁰ MEL (mouse) or LAM-1 (human)). See, Siegellman et al., Science, 243: 1165-1172 (1989); Rosen, Cell Biology, 1: 913-919 (1989) and Love et al. Cell, 56: 1045-1055 (1989) as incorporated herein by reference. In addition to lymphocyte capture, LHR is thought to act initially in inflammatory reactions and mediate neutrophil binding to endothelium.

The structure and function of the lactin receptors has been elucidated by cloning and expressing full-length cDNA for each of the above receptors (see, e.g., Bevilacqua et al., Science, supra, (ELAM-1), Geng et al., Supra (GMP 140) , and Love et al., supra (MEL-14)). The extacellular portion of the selectins can be divided into three segments according to the homologies to the proteins described above. The amino terminal region (about 120 amino acids) is related to a family of mammalian C-type lectin proteins that includes the low affinity IgE receptor CD23. Residues 121-155 are related to proteins containing the [motif] epidermal growth factor (EGF) motif. The EGF domain is followed by 3 to 6 tandem repetitive motifs, related to those found in complement regulatory proteins.

The immunoglobulins of the invention recognize and selectively bind functional epitopes to ELAM-1 and thus inhibit intercellular adhesion in in vitro assays. The immunoglobulins described herein as examples can be used in various standard screening procedures to identify additional immunoglobulins within the scope of the invention. In addition, the in vivo evidence provided herein demonstrates that the claimed antibodies are also effective in treating inflammatory conditions mediated by ELAM-1. A functional epitope as used herein is an antigenic site on ELAM-1 that is selectively bound by an antibody that substantially inhibits the binding of the ELAM-1 ligand to the ELAM-1 receptor and thereby inhibits a disease inflammatory response in a patient. For the purposes of this application, the inhibition is at least about 60%, preferably about 70% to 90%, and more often about 99% or more (as measured in an in vitro assay as described below).

A number of antibodies were identified by screening for activated endothelial cells. See e.g. Bevilacqua et al., Proc. Natl. Acad. Sci., Supra; Pober et al., J. Immunol. 136: 1680 (1986); Graber et al., J. Immunol., 1.45: 819-830 (1990); Leeuwenberg et al., J. Immunol., 156: 2110-2114 (1990); Wellicome et al., J. Immunol., 144: 2558-2568 (1990); and PCT Publications no. WO 90/05768, WO 90/05539 and WO 90/13300, as incorporated herein by reference.

The claimed immunoglobulins are suitable. modification using a wide variety of techniques available to those skilled in the production and manipulation of various immunoglobulin molecules. Several possible modifications are discussed in more detail below.

The basic structural unit of an immunoglobulin is formed by a known tetrmer. Each teramer consists of two identical pairs of polypeptide chains, and each pair has one light (about 25 kD) and one heavy chain (about 50-70 kD). The amino end of each chain determines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy terminus of each chain determines the constant region primarily responsible for effector function.

The term immunoglobulin as used herein refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Identified immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu [mu] constant region genes as well as tens of thousands [myriad] variable region genes. The light chains are sorted into kappa and lambda. Heavy chains are sorted into gamma, mi, alpha, delta, or epsilon, which then defines the respective classes of immunoglobulins, IgG, IgM, IgA, IgD, and IgE.

Immunoglobulins can, in addition to complete antibodies, exist in red of various forms, including, for example, Fv, Fab, F (ab ') ₂ , as well as single-stranded forms (e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988), Bird et al.

Science, 242: 423-426 (1988), and Hunkapiller and Hood, Nature, 323:

15-16 (1986), as incorporated herein by reference).

Treatment with the protease papain, the molecule is cleaved into three fragments, two of which are indicated Fab fragments and one _Fc fragment. Each Fab fragment consists of an antigen binding domain and a C _H 1 domain. Further proteolytic processing of Fab fragments releases an Fv fragment that contains only the variable region. The protease pepsin cleaves the heavy chain at amino acid residues 234 and 233 to yield F (ab ') ₂ and pFc' fragments.

The F _c fragment, which consists of the C _H 3 domains, is that part of the immunoglobulin molecule that mediates various effector functions. Depending on the heavy chains in immunoglobulins, there are a number of different effector functions. They include complement fixation, B cell stimulation, persistence in circulation, and binding to F _c receptors on granulocytes and macrophages prior to phagocytosis. See, generally, Fundamental Immunology, 2nd Edition, WE Paul, ed., Ravens Press NY (1989), and Winkelhake, Immunochem., 15: 695-714 (1978), as incorporated herein by reference.

The immunoglobulins of the invention can be produced in a number of different ways. Production of non-human monoclonal antibodies, e.g. mouse, rodent, horse, etc. is well known and can be performed, for example, by immunizing an animal with an isolated ELAM-1 receptor, activated by endothelial cells, or cells transformed with DNA encoding the ELAM-1 receptor. Antibody-producing cells obtained from immunized animals are immortalized and are screened, or first evaluated for the production of the desired antibody and then immortalized.For an analysis of general procedures for producing monoclonal antibodies, see Harlow and Lane, Antibodies, A Laboratory Manual (1988) For methods suitable for generating antibodies to ELAM-1, see Bevilacqua et al., Proc. Natl. Acad. Sci., supra;

Pober et al., J. Immunol. 136: 1680 (1986); Graber et al., J. Immunol., 145: 819-830 (1990); Leeuwenberg et al., J. Immunol., 156: 2110-2114 (1990); Wellicome et al., J. Immunol., 144: 2558-2568 (1990); EP Publication No. 408,859 A2; and PCT Publications no. WO 90/05768, WO 90/05539 and WO 90/13300, as incorporated herein by reference.

The monoclonal antibodies of the invention are preferably evaluated for their ability to inhibit the adhesion mediated by ELAM-1 and other selectin receptors in the assays as described in Example 1 below. The assays, ideally, allow the screening and evaluation of immunoglobulins on a large in vitro scale. Many direct and indirect methods for in vitro screening for inhibitors of ligand-receptor interactions are available and known to those skilled in the art. Example: The ability to inhibit the adhesion of ligand-bearing cells such as PMN to cells expressing a particular selectin can be determined. As discussed above, selectin receptor genes have been cloned, and so these genes can be introduced and expressed in a wide variety of different cells, such as COS cells, CHO cells, and the like. Typically, the test immunoglobulin is incubated with labeled ligand-bearing cells and activated selectin-bearing cells that are immobilized on a solid surface. The inhibition of cell adhesion is then determined by detecting the label bound to the surface after appropriate washing. In the example assay described below, PMN cells and activated human endothelial cells or activated platelets were used.

The immunoglobulins identified by the assays described above are suitable for a variety of pharmaceutical, diagnostic and other uses. In addition to the treatment and diagnosis of selectin-mediated diseases, fully described below, immunoglobulins can be used to screen for other monoclonal antibodies, which recognize a functional epitope. Such urine antibodies are identified by their ability to block binding of the claimed immunoglobulins to cells expressing a selectin receptor.

However, the therapeutic efficacy in humans is often limited with respect to non-human antibodies because they have a short serum half-life and elicit human immune responses. Thus, it may be desirable to modify the antibodies for better therapeutic use. A variety of strategies are available to enhance diagnosis and therapy using monoclonal antibodies, see Waldmann, Sience 252: 1657-1662 (1991), as incorporated herein by reference.

One method suitable for modifying the antibodies of the invention is to transfer an antigen binding region from non-human antibodies to portions of human antibodies. For example, the F (ab ') ₂ fragment can be attached to a human constant region to form chimeric antibodies. Alternatively, hypervariable regions can be attached to framework human regions to create what is termed humanized antibodies herein. Such methods are generally known in the art and are described, for example, in US-US. No. 4,816,397, U.S. Pat. No. 4,816,567, and EP publications 173,494 and 239,400, PCT publication no. WO / 90/07861 and Reichmann, L. et al., Nature, 332: 323-327 (1988), as incorporated herein by reference.

Chimeric and humanized antibodies are typically constructed by recombinant DNA techniques from gene segments belonging to different species. For example: The variable (V) segment of the mouse immunoglobulin gene may associate with a human constant segment, such as a τ ₃ · A preferred therapeutic chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain of a mouse antibody and the C or effector domain of human antibodies, although other mammalian species may be used.

Chimeric and humanized antibodies have a number of potential advantages over murine antibodies when used in human therapy. For example: The human immune system may not recognize the C regions of the chimeric antibodies as foreign and therefore the antibody response against the injected chimeric antibody will be less than against the total foreign mouse antibody. In addition, injectable murine antibodies are said to have a much shorter half-life in human circulation than human antibodies of the same class (Shaw et al., J. Immunol., 138: 4534-4538 (1987)). administered chimeric antibodies will have a half-life closer to human antibodies and allow smaller and less frequent doses to be given.

Another method for producing antibodies of the invention is to isolate DNA sequences that encode a human monoclonal antibody to a human or a selectin portion thereof in a library

DNA from human which specifically binds to the receptor, by screening for B cells according to the general protocol outlined in Husa et al., Science, 246: 1275-1281 (1989), as incorporated herein by reference, and then cloning and amplifying the sequence, which encodes an antibody (or binding fragment) with the desired specificity.

In one aspect, the invention is directed to regions of recombinant DNA encoding the variable or hypervariable regions of the light and / or heavy chains of the claimed monoclonal antibodies. DNA regions encoding these regions are typically joined to DNA regions encoding appropriate constant regions, such as human gamma heavy chain regions or human lambda light chain regions. Those skilled in the art will recognize that, for codon degeneration and non-critical amino acid substitutions, these sequences may be readily replaced by sequences other than those discussed below.

The DNA regions will typically further comprise expression control DNA sequences operably linked to the coding sequences of the chimeric antibodies, including naturally relevant or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, host! is maintained under conditions suitable for high level expression of nucleotide sequences, and may be followed, as desired, by the collection and purification of light chains, heavy chains, dimers of light and heavy chains, or intact antibodies.

It is well known that natural immunoglobulins will differ somewhat as a result of deletions, substitutions, insertions of maturation lengths, because of the addition of one or more amino acids in a given sequence. Thus, both variable and constant regions are subject to substantial natural modification, but are still substantially identical and still retain their respective activities. Human constant region DNA sequences can be isolated according to well known procedures from a variety of human cells, but preferably from immortalized B cells or long-term human B cells (Banchereau, et al., Science, 251: 70-72 (1991), as described above). here by reference). A suitable source cell for DNA sequences and host cells for expression and secretion can be obtained from numerous sources, such as the American Type Culture Collection (1985 edition of Cell Lines and Hybridomas, Fifth edition, Rockville, Maryland, USA, incorporated herein by reference). ).

In addition to these naturally occurring forms of immunoglobulin chains, substantially identical modified heavy and light chains can be readily knocked out and made using recombinant DNA techniques well known to those skilled in the art. For example: The chains may differ from the naturally occurring sequence at the level of the primary structure by several amino acid substitutions, terminal and internal additions and deletions, and the like. Alternatively, polypeptide fragments containing only a portion (usually at least about 60-80%, of the structure) can be produced, and these fragments typically have 90-95

%) primary or more immunoglobulin activities (e.g., complement) but exhibiting lower activity in immunogenicity.

fixation

In particular, it is believed that, like many genes, (i) the genes belonging to immunoglobulins contain separate functional regions, each corresponding to one or more distinct biological activities. These regions can be fused to functional regions from other genes (e.g., enzymes) to form fusion proteins (e.g., immunotoxins) that have novel properties. In general, modification of these genes can be performed by a variety of well known techniques, such as targeted mutagenesis (see Gillman and Smith, Gene, 8: 81-97 (1979) and Roberts, S. et al., Nature, 328: 731-734 ( 1987), as incorporated herein by reference).

Nucleic acid sequences of the invention capable of ultimately expressing the desired chimeric antibody can be generated from a variety of polynucleotides (genomic or cDNA, RNA, etc.) and components (e.g., V, J, D, and C regions), as well as a variety of techniques . Linking of the appropriate gene sequences is currently the most common method of production, but cDNA sequences can also be used (European Patent Application Nos. 85102655.S,

85305604.2, 84302356.0 and 85115311.4 as well as PCT Applications Nos. GB85 / 00392 and US86 / 02269, as incorporated herein by reference).

As previously stated, the DNA sequences will be expressed in the host upon practical attachment (i.e., a placement that guarantees functioning) expression vectors are organisms, either to sequences that are typically replicable as episomes or as expression. These in the host integral part of the host chromosomal DNA. Typically, expression vectors will contain a selectable marker, e.g. tetracycline or neomycin to allow detection of those cells that are transformed with the desired DNA sequences (see, e.g., U.S. Patent 4,704,36 2, as incorporated herein by reference).

E. coli is one pro-prokaryotic host, particularly useful for cloning the DNA sequences of the invention. Other microbial hosts suitable for use are Bacillus subtilis, other enterobacteria such as Salmonella and Serratia, and various Pseudomonas species. Expression vectors can also be prepared in these prokaryotic hosts, typically containing expression control sequences compatible with the host cell (e.g., origin of replication). In addition, any number of well-known promoters, such as the lactose promoter system, the tryptophan promoter system (trp), the beta-lactamase promoter system, or the lambda phage promoter system will be present. Promoters will typically drive expression, optionally with an operator sequence, and will have ribosome binding site sequences and the like to initiate and complete transcription and translation.

Other microbes, such as yeast, can also be used for expression. Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzyme, origins of replication, termination sequences, and the like, as desired.

In addition to microorganisms, mammalian tissue culture cells can also be used to produce the polypeptides of the invention {see Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987), as incorporated herein by reference). Eukaryotic cells are in fact preferred because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell line, various COS cell lines, HeLa cells, myeloma cell lines, etc., but preferably transformed B cells and hybridomas. Expression vectors for these cells may include expression control sequences, such as. They are. origin, replication, promoter, enhancer (Queen, C. et al., Immunol. Rev., 89: 49-68 (1986), as incorporated herein by reference), and necessary information processing sites such as ribosome binding site , an RNA splicing site, a polyadenylation site, and transcriptional termination sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, and the like.

Vectors containing regions of DNA of interest (e.g., heavy and light chain coding and expression control sequences) can be transferred to host cells by well known methods, varying depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment can be used for other cell hosts. See, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, (1989), as incorporated herein by reference.

Once the whole chimeric antibodies, their dimers are expressed, since the individual light and heavy chains of the invention can be purified by standard procedures in the art, including ammonium sulfate precipitation, fractional column chromatography, gel electrophoresis, and the like (see, generally,

Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982)). Once purified, partially or to homogeneity as desired, the polypeptides can be used therapeutically or for the development and performance of analytical procedures, immunofluorescent staining and the like (see, generally, Immunological Methods, Vol. I and II, ed. Lefkovits and Pernis, Academic Press, New York, NY (1979 and 1981).

The compositions of the invention contain monoclonal antibodies that selectively bind to selectin receptors on cells in conjunction with numerous disorders. For example: Numerous inflammatory disorders are associated with selectins expressed on vascular endothelial cells. The term inflammation is used to refer to the responses of both a specific and a non-specific defense system. The response of a specific defense system is the response of a specific immune system. on .... antigen. ... An example of a specific defense system response includes an antibody response to antigens such as viruses and hypersensitivity of the subsequent type. The response of a non-specific defense system is an inflammatory response mediated by leukocytes, generally incapable of immunological memory. Such cells include granulocytes and macrophages. Examples of non-specific reactions include collecting PMN leukocytes at sites of bacterial infection (eg, pulmonary infiltrates in bacterial pneumons and pus formation in abscesses).

Inflammatory conditions treatable according to the invention include e.g. septic shock, wound-associated sepsis, rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), acute leukocyte-mediated lung injury (e.g., post-stress respiratory syndrome of the elderly), immune-mediated tissue damage (e.g. , including atopic dermatitis and psoriasis. In addition, tumor metastasis can be prevented by inhibiting the adhesion of circulating tumor cells. Examples include colon carcinoma and melanomas.

The antibodies and pharmaceutical compositions thereof of the invention are particularly useful for parenteral administration, i.

subcutaneous, intramuscular and inravenous. Intact immunoglobulins or binding fragments thereof such as Fab, F (ab ') _2, etc. are suitable for use in pharmaceutical compositions. In addition, a number of new drug delivery approaches are under development, and the pharmaceutical compositions of this invention are also suitable for administration using these new methods. See Langer, Science,

249: 1527-1533 (1990) as incorporated herein by reference.

In one example, the antibodies of the invention can be used to target conventional anti-inflammatory drugs to specific tissue damage sites. By using antibodies to target the drug to ELAM-1, such drug may achieve a higher concentration at the site of injury. The side effects of conventional anti-inflammatory chemotherapeutic agents can be substantially alleviated by lower doses, localization of the agent at the site of injury. and / or by encapsulating the agent prior to administration. The antibodies may be directly or indirectly coupled to a chemotherapeutic agent. The junction that can be made by means generally known in the art must not substantially inhibit the ability of the antibody to bind the receptor, nor must it significantly reduce the activity of the chemotherapeutic agent. A number of different chemotherapeutic agents can be attached for targeting. For example: Anti-inflammatory agents that can be attached include immunomodulators, platelet activating factor antagonists,

PAF), cyclooxygenase inhibitors, lipoxygenase inhibitors, and leukotriene antagonists. Preferred moieties include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, and the like. Similarly, antioxidants, e.g. superoxide dismutase, are useful for treating reperfusion injury. Similarly, anti-tumor agents such as daunomycin, doxorubicin, vinblastine, bleomycin, etc. can be targeted.

Targeting to the selectin receptor can also be achieved with amphipaths, i.e. molecules with a double character (polar / non-polar) which exist as aggregates in aqueous solutions. Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithins, phospholipids, saponins, bile acids and soles. These molecules may exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are generically referred to herein as liposomes. In such compositions, the drug to be administered is part of a liposome together with the antibody of the invention. When the liposomes come close to the affected cells, they deliver the selected therapeutic agent

The liposomes of the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol such as cholesterol. The choice of lipids is governed by e.g. the size of the liposomes and the stability of the liposomes in the bloodstream.

Liposome targeting using a variety of targeting agents (e.g., ligands, receptors, and monoclonal antibodies) is well known in the art. 4, -957,773 and 4,603,044, as incorporated herein by reference). Standard methods for linking targeting agents and liposomes can be used. Liposomes targeted by antibodies can be constructed using, for example, liposomes containing protein A. {See Renneisen et al., J. Biol. Chem., 265: 16337-16342 (1990) and Leonetti et al., Proc. Natl. Acad. Sci. (USA) 87: 2448-2451 (1990), as incorporated herein by reference).

Liposome charge is an important determinant in the purification of blood from liposomes, negatively. charged, they are taken up by the reticuloendothelial system more quickly (Juliano, Biochem. Biophys. Res. Commun. 63: 651 (1975)) and thus have a shorter half-life in the bloodstream. Liposomes with prolonged circulating half-life are typically desirable for therapeutic and diagnostic uses. Liposomes that can be maintained from 8, 12 or up to 24 hours in the bloodstream provide sustained release of the anti-inflammatory factor. Serum half-life may also be important if liposomes or antibodies are labeled for in vivo diagnostic imaging.

A variety of methods are available for the preparation of liposomes, as described e.g. in Szoka et al., Ann. Rev. Biophys. Bioeng. , 9: 467 (1980), US Patent NOS. 4,235,871, 4,501,728, and 4,837,028, as incorporated herein by reference. One method creates multilamellar vesicles, heterogeneous in size. In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system, and dried under vacuum or under inert gas to form a thin lipid film. If desired, the film may be redissolved in a suitable solvent, such as tertiary butyl alcohol, and then lyophilized to form a more homogeneous lipid mixture that is in a more easily hydratable form similar to a powder. This film is coated with an aqueous solution of the drug and the targeting component (antibody) and allowed to hydrate, typically over 15-60 minutes, with agitation. Size distribution of the resulting multilamellar vesicles can be shifted to smaller sizes in terms of intensive shaking because adding solubilizing detergents such as deoxycholate _...... .... ...........

The hydration medium contains the targeted drug at a concentration that is desirable in the internal volume of the liposomes in their final suspension. Typically, the drug solution contains 10-100 mg / ml in a buffered salt. The antibody concentration is generally between about 0.1-20 mg / ml.

Pushing liposomes through a small pore polycarbonate membrane or asymmetric ceramic membrane is also an effective method to reduce the size of the liposomes and to achieve a relatively well defined size distribution. Typically, the suspension circulates once or more once through the membrane until the desired liposome size distribution is achieved. Liposomes can be pushed through membranes with stepped smaller pores to achieve a gradual reduction in liposome size.

Even with the most efficient encapsulation methods, the original, sized, liposomes can contain up to 50% or more of the drug and targeting agent in free (unencapsulated) form. For this reason, in order to maximize the benefits of liposomal drug targeting, it is important to remove the free drug and targeting agent from the final suspension suitable for injection. Several methods are available to remove non-trapped substances from a liposome suspension. In one method, liposomes from the suspension are deposited by high-speed centrifugation and free substances and very small liposomes remain in the supernatant. Another method involves concentrating the liposomes by ultrafiltration, and the concentrated liposomes are then resuspended in a drug-free replacement medium. Alternatively, gel filtration can be used to separate large liposomes from molecules in solution.

After treatment to remove free drug and / or targeting agent, the liposome suspension is adjusted to the desired concentration for intravenous administration. This may include resuspending the liposomes in a suitable volume of the incubation medium in which the liposomes have been concentrated, for example by centrifugation or ultrafiltration, or concentrating the suspension if the drug removal step has increased the total volume of the suspension. The suspension is then sterilized by filtration. Liposome-ligand preparations can then be administered as described below.

As discussed above, pharmaceutical compositions (comprising liposomes or free antibodies) are particularly useful for parenteral antibodies or administration. Typically, the composition comprises a cocktail solution thereof, dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of carriers are useful, e.g. water, buffered water, 0.4% salt, 0.3% glycine and the like. These solutions are sterile and generally do not contain particulate material. These compositions can be sterilized by conventional, well known techniques. The compositions may contain pharmaceutically acceptable minor excipients which are required to approximate physiological conditions, such as pH adjusters and buffering agents, to adjust tonicity (e.g.

isotonic) and the like, for example, potassium chloride, calcium chloride, sodium, sodium chloride, sodium lactate, etc.

The antibody concentration in these formulations can be varied over a wide range, from less than 0.5%, through the usual 1% or at least 1%, to as high as 15 to 20% by weight, and is selected primarily on the basis of liquid volumes, viscosities, etc., according to the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intramuscular infections can be prepared by containing 1 ml of sterile buffered water and 50 mg of antibody. A typical composition for intravenous infusion may be prepared by containing 250 ml of sterile Ringer's solution and 150 mg of antibody. Particular methods for preparing parenterally administered compositions will be known or clear to those skilled in the art and are described in more detail in e.g. Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pennsylvania (1985), as incorporated herein by reference.

The antibodies of the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and lyophilization and reconstitution techniques, as known in the art, can be utilized. Those skilled in the art will recognize that lyophilization and reconstitution can lead; at varying degrees, to loss of antibody activity (e.g., with conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies) and that the levels applied should be adjusted for compensation.

Compositions containing the disclosed antibodies or cocktails therefrom can be administered for prophylactic and / or therapeutic effects. patient

When administered therapeutically, in sufficient amounts they are administered to cure or at least partially reduce the infection or its complications. An amount adequate to achieve this is defined as a therapeutically effective dose. The amounts effective in such use will depend on the severity of the disease and the general condition of the patient's own immune system, but will generally vary between about 1 mg / kg body weight to about 20 mg / kg body weight, preferably between about 5 mg / kg. kg body weight to about 15 mg / kg body weight. It is to be understood that the materials of the present invention can generally be used in serious disease states, i.e., life-threatening or potentially life-threatening situations. In such cases, bearing in mind the minimization of foreign matter and the low likelihood of rejection of foreign substances that is achieved by the human chimeric antibodies of the present invention, a substantial excess of these antibodies can and will be considered desirable by the attending physician.

For prophylactic administration, compositions containing the disclosed antibodies or cocktails therefrom are administered to a patient who is not yet in a disease state to increase his resistance. Such an amount is defined as a prophylactic effective dose. In such use, the precise amounts are again dependent on the patient's condition and health and the general level of immunity, but are generally within the range described above.

Single or repeated administration of the compositions may take place at a dosage level and with a pattern selected by the attending physician. In any event, the pharmaceutical compositions are intended to provide an amount of the antibody (s) of the invention sufficient to effectively treat the patient. The antibodies of the invention can also be used for diagnostic purposes such as determining the area of inflammation. For diagnostic purposes, antibodies can be either labeled or unlabeled. Unlabeled antibodies can be used in combination with other, labeled antibodies (second antibodies) that react with a given antibody, such as antibodies specific for a particular immunoglobulin constant region. Alternatively, the antibodies may be directly labeled. A wide variety of labels, such as radionuclides, fluorescent compounds, enzymes, enzyme substrates, enzyme cofactors, ligands (preferably haptens), etc., can be used. Numerous types of immunoassays are available and well known to those skilled in the art.

For prophylactic administration, preparations containing antibodies or shakes thereof are administered to a patient suspected of having an inflammatory disease condition. Alternatively, the efficacy of a particular treatment can be monitored. The amount required for this is defined as a diagnostically effective dose. In this use, the exact amounts will depend on the patient's condition and health and the like.

Kits with pre-term antibodies can also be supplied. Thus, compositions of the subject antibody can be provided, usually in lyophilized form and in a reservoir, alone or in combination with other antibodies specific to the desired cell type. Antibodies that can be conjugated to a label or toxin or with buffers such as Tris, stabilizers, biocides, unconjugated, include the kit phosphate, carbonate, and the like, inert proteins, e.g.

serum albumin, and a set of instructions for use. Generally, the material additions will be present in less than 5% by weight, calculated on the active antibody, and usually in a total amount of

0.001% by weight, calculated again for antibody concentration. It will often be desirable to include an inert volume enhancer or excipient to dilute the active ingredients, wherein the excipient may be present in the form of 1-99% by weight of the total composition. If a second antibody capable of binding to a chimeric antibody is used in the assay, it will usually be contained in a separate vial. The second antibody is typically conjugated to a label and formulated in a manner similar to the antibody formulations described above.

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

Example 1

This example demonstrates the ability of the monoclonal antibodies of the invention to block neutrophil (PMN) adhesion to activated vascular endothelial cells in the determination of intracellular adhesion. The determination procedure was as follows:

human umbilical cords were obtained after delivery. They were 20-40 cm long and without traces of ligation. Under sterile conditions, one end of the vein part of the umbilical cord was cannulated and connected to a 3-way cock and an injection syringe. The cord was washed with phosphate buffered saline (PBS) containing no Ca + / Mg + to remove blood and clots. The opposite end of the cord was then cannulated and attached to the two-way cock; followed by a short collagenase flush (Boehringer Mannheim) of 1 mg / ml and the two-way cock was closed. The cord was then filled with collagenase, air was removed, and the 3-way cock was closed. The cord was placed in a sterile chamber and incubated at 37 ° C for 15-20 minutes. After incubation, the string was washed with 30 ml PBS containing Ca ⁺⁺ and Mg ⁺⁺ and the eluted endothelial cells were collected in a sterile tube containing 5 ml EGM-UV medium (Clonetics) and 10% FCS to inhibit further collagenase action. Cells were centrifuged and resuspended in complete medium containing EGM-UV, 10 ng / ml epidermal growth factor, 1 µg / ml hydrocortisone, Gentamicin, Amphotericin-B, and 10% FCS. A tissue culture flask of appropriate size (depending on the length of the strand) was covered with 0.1% gelatin (endotoxin-free, isolated from bovine epidermis) for 15 min. at room temperature and the excess was removed. The endothelial cell suspension was transferred to a bottle and placed in a 5% CO ₂ incubator. The medium was changed after 16 hours when the cells seemed to adhere. Cells were passaged prior to confluency (75%). For cell passage, the medium was aspirated under vacuum, the bottle was washed two times with HEPES (10 mM) buffered salt to remove FCS, and then 1-2 ml of 0.025% trypsin EDTA was added to the bottle and examined by inverting microscope until endothelial cells reached suspension (30 sec to 1 min), fresh complete medium was then added to the bottle and dispensed 1: 2 or 1: 3 into new gelatin-coated bottles.

To form endothelial cell monolayers on 96-well assay plates, the plates were coated with gelatin as described above. Endothelial cells that were passaged 3 times were harvested from flasks using 0.025% trypsin as above and plated at 5 x 10 ⁴ cells per well. Cells were allowed to grow to confluency. The medium was changed once applied.

Neutrophils (PMN) were prepared from whole blood. 50 ml of blood was collected from a voluntary donor into heparinized vacutainer tubes. Each 25 ml of blood was layered over 15 ml of Mono-Poly Resolving Medium (Flow Labs) .The tubes were centrifuged at 20 ° C for 25 minutes at 2000 rpm in an RT6000 centrifuge and then the speed was increased to 2,500 revolutions per minute for an additional 25 minutes The PNM layer (the lower of the two hovering cell layers) was transferred to a clean 50 cm ³ centrifuge tube, and 30 ml of Hank's Balanced Salt Solution was added to each tube. (HBBS) (Gibco) containing 20 mM HEPES (Gibco) and 0.2% glucose (Fisher) The tubes were then centrifuged at 3,000 rpm for 3 minutes at room temperature and resuspended in HBSS / HEPES / glucose buffer three times. and calculated using a hemacytometer.

To perform the assays, assay plates were removed from the incubator and the adherent cells were washed twice with HBSS / HEPES / glucose + 5 mg / ml bovine serum albumin. 100 μΐ of each monoclonal antibody tested was added to the wells of the assay plate. Antibodies were incubated on the plate for 20 minutes. 5 x 10 ⁵ PMN was added to the wells v.50 μΐ. The plates were then incubated for 6 minutes at room temperature. Non-adherent cells were removed from the wells by inverting the plate over the sink and adding 200 μΐ medium using a multi-channel pipette four times. The last wash liquid was removed from the wells and 50 µL of solubilization buffer was added. This consisted of citrate buffer (24.3 ml of 0.1 M citric acid,

10.5 g / 500 ml + 25.7 ml 0.2 M sodium hydrogen phosphate, 1.42 g / 500 ml and SQ H ₂ to 100 ml) containing 0.1% NP-40 detergent. The plate was incubated with gentle agitation for 10 minutes and then 50 μΐ OPDA (8 mg o-phenylenediamine, Sigma Cat # P-1526, 8 μΐ 30% H ₂ O ₂ and 10 ml SQ H ₂ 0) was added to each well. . The plates were incubated for 15 minutes at room temperature and then 15 μΐ 4 MH ₂ SO ₄ was added to each well to stop the reaction. Control reagent mix was prepared by mixing 50 μΐ of solubilization buffer, 50 μΐ of OPDA solution and μΐ 4 transferred

MH ₂ SO ₄ 100 μΐ was removed from each well and into a flexible ELISA assay plate (Falcon).

The plate was scanned at 492 nm within 30 minutes.

In addition, colo 205 carcinoma cells (ATCC No. CCL 22) known to express SLX were used. Since these cells do not carry the F _c receptor, there should be no cross-linking by monoclonal antibodies. The assay procedure was the same as described above for PMN. However, the cells were labeled with ⁵¹ Cr (50 μΟί / ^υη ^ after 30 min). Adhesion was determined using a gamma counter and standard procedures.

The results presented in Table 1 below show that the monoclonal antibodies of the invention effectively block the adhesion of PMN and Colo 205 cells to activated endothelial cells. The prior art monoclonal antibody, H18 / 7 (described in PCT Publication No. WO 90/05539, supra) was included as a control. Binding quantity in the absence of antibodies was arbitrarily assigned a value of 1.0. Antibodies that promote binding in the assay have a value greater than 1, while antibodies that inhibit binding have a value less than 1.

Table 1

Binding of cells to HUVEC in the presence and absence of antibodies to ELAM-1

Mab Subclass PMN Carcinoma (Colo 205)

any Ig _G1 1.00 1.00 EB1-5 IgG, 1.42 0.08 ENB1-6 IgG, 1.64 0.76 EB1-7 IgG _x 1.48 0.10 EB3-1 IgG ₃ 0.18 0.12 EB3-2 IgG ₃ 0.3 0.12 EBM-2 IgG _x 0.21 0.12 EBM-4 IgG _x 0.28 0.10 H18 / 7 IgG ₂ ; or b 1.20 0.16

Example 2

This example presents competitive inhibition assay data used to compare the epitope recognized by the monoclonal antibodies of the invention to the epitope recognized by the mononoclonal antibody first identified as binding ELAM-1 (H18 / 7). To do this, H18 / 7 was biotinylated and HRP-avidin staining was detected by solid-phase ELISA on IL-1 activated entothelial cells, according to standard procedures (see, e.g., Harlow and Lane, supra). The values given are absrobances at 492 nm.

As shown in Table 2, some inhibition of H18 / 7 binding was found.

Table 2

Blocking Mab Biotinylated Mab HRP-avidin inspection H18 / 7 1,175 H18 / 7 H18 / 7 0,309 EB1-1 H18 / 7 1,021 EB3-1 H18 / 7 0,830 ENB1-6 H18 / 7 0,346

Example 3

This example demonstrates the ability of the monoclonal antibodies of the invention to block neutrophil (PMN) adhesion to activated platelets (carrying GMP-140) in the intercellular assay. The results of such assays are presented in Table 3, which shows the relative adhesion of PMN or Colo 205 cells to activated platelets in the presence of these monoclonal antibodies of the invention. The determination procedure was as follows:

Plates were obtained from a normal human donor and were washed from plasma proteins in the presence of PGE-1 (100 nM) (see Polley and Nachman, J. Exp. Med., 158: 603 (1983) as incorporated herein by reference). They were activated by thrombin (0.25 U / ml; 2 x 10 6 plates per ml) for 20 minutes at room temperature without stirring.

Two assays were used to determine GMP-140-mediated adhesion to activated platelets: assay plate assay and liquid phase assay. Assay on assay plates is a modification of the endothelial cell and neutrophil adherence assay described previously (Dobrina et al., Immunology, 158: 502 (1989) as incorporated herein by reference). Platelet suspension (300 μΐ, 10 ⁸ / ml) was placed in each well of a 48-well assay plate that was pre-coated with 0.1% gelatin. Plate was. incubated for 15 minutes at room temperature. 37 ° C, then centered for 2 minutes at 90 xg and washed twice with phosphate buffered saline (PBS) to remove non-adherent plates. To block the sufficient F _c receptor, 300 μΐ of heat-aggregated IgG (20 μΐ / ml) was added to each well and the plate was allowed to stand at room temperature for 20 minutes. The plate was washed once with PBS and then 300 μΐ of monoclonal antibody was added, which was tested, and the plate was allowed to stand at room temperature for another 20 minutes. Meanwhile, neutrophils were isolated from whole human blood by the method described above. They were radiolabeled ^x Cr (450 µCi added to 3 x 10 ⁶ cells at 300 µΐ and incubated for 60 minutes at 37 ° C), washed three times with RPMI containing 10% fetal calf serum, and resuspended at 2 x 10 ⁸ / ml . 50 μΐ of this suspension was added to each well of the assay plate. The plate was centrifuged for 2 minutes at 90 xg and then allowed to stand for 5 minutes at room temperature. Unbound cells were removed by washing three times with PBS and the remaining bound cells were released from the plate using a buffer containing SDS and then processed to measure radioactivity.

The liquid phase assay was performed by modification of the described (see Larsen et al., Cell, 63: 467 (1990) as incorporated herein by reference). 20 μΐ of activated platelets (2 x 10 / ml) were placed in an Eppedorf tube. 20 μΐ of heat inactivated IgG was added and after mixing the tubes were allowed to stand at room temperature for 20 minutes. 20 μΐ monoclonal antibody was added and the tubes were allowed to stand for another 20 minutes at room temperature. Meanwhile, PMN was prepared as above (without radiolabeling) and diluted to 2 x 10 ° / ml. 20 μΐ of this suspension was added to each tube and the tubes were allowed to stand for 20 minutes at room temperature. Adhesion was evaluated microscopically and was expressed as the percentage of cells tested that bound two or more platelets.

In addition, assays were performed using cells derived from colon carcinoma, Colo 205 (ATCC No. CCL 222), which are known to express a ligand recognized by a selectin receptor. The assay procedure was the same as that used with PMN. ............................... ................... ..... ......... ...................... ............ .... .. ....

Table 3

precipitates

MAb PMN Carcinoma (Colo 205) purified GMP-140 any + + • · · GBI-1 - - + GBI-2 - - + GNBI-3 + + + Example 4

This example shows the efficacy of mAb EB3-1 in an animal model of liposaccharide-induced death. The rat system was selected because EB3-1 was shown to cross-react with rat ELAM-1 equivalent.

LPS from E. coli 0111: B4 (Sigma, Lot # 36F4019) were freshly prepared from a single lot, one day before use, by dissolution in sterile, pyrogen-free, saline at a concentration of 5 mg / ml. The solution was sonicated on ice for 30 seconds using a Tekmark Sonic disintegrator. Just before use, the solution was sonicated a second time for 30 seconds.

Female Lewis rats weighing 200 g (± 10 g) were obtained from Charles River Breeding Labs and kept for at least 7 days after delivery (for adaptation). Groups of 10 animals were used unless otherwise stated. All reagents were injected parenterally, via the tail vein and 0.5-1.0 ml / kg. As a negative control, animals received either sterile LPS-free saline, as murine IgG3k myeloma protein (J606, low pyrogen - <2 ng / mg protein).

The EB3-1 dose and schedule protocol was empirically derived from the pharmacokinetic data obtained with EB3-1 administered to the rat prophylactically. The minimum LD ₁₀₀ dose of LPS for these rats was determined to be 7.5 mg / Kg.

In one experiment, rats were given 10 mg / kg of P6E2 one hour before the attack ...... [chalenge] .LPS. . Amplify dose… [a. bo.ost. previously, it was administered three hours after the attack (1.0 mg / kg). 4/10 of the animals survived the LPS attack. In contrast, all 10 controls (which received a salt solution infection) died. During the 24-hour observation period, surviving animals had few clinical signs characteristic of animals treated with LPS.

We then tested doses of EB3-1 that were (1) two times higher and (2) orders of magnitude lower than the 10 mg / kg dose used in the first experiment. The results show that EB3-1 has a significant effect: 80% of the animals survived at 10 mg / kg (Fig. 1). Note that one animal survived in the control group receiving the salt solution infection - this suggests that the group of animals was not affected by the LPS attack as hard as the animals in the previous experiment.

Another study was performed to show the therapeutic value of EB3-1. Animals received an iv bolus dose of 10 mg / kg 1 hour before or 2, 4, or 6 hours after LPS attack.

Again this time, 1 in 10 animals receiving saline survived as well as from the group receiving 10 mg / kg of J606 myeloma protein at T = -60 minutes (Fig. 2). However, EB3-1 had a significant protective effect even when administered 2 or hours after LPS.

conclusion

The protection observed with Cytel mAb EB3-1 shows that the antibodies of the invention are useful as therapeutically in the treatment of inflammatory mediated ELAM-1.

prophylactic as well as disease reactions

Example 5

This example demonstrates the efficacy of purified EB3-1 antibody in inhibiting lipotechoic acid (LTA) inflammatory response in rat pleural cavity. The ability of LTA to induce pleuritis was determined with linear dose concentration and neutrophil infiltration. LTA has also been shown to activate endothelium through ELAM-1 to neutrophil adhesion in vitro (see Example 6, below).

In this experiment, we induced pleuritis in rats by bilateral LTA infection into the pleural cavity. In the test animals, the mAb was injected into the tail vein. The cavities were flushed after 4 hours and the evaluation was performed according to the neutrophil concentration in the exudates.

Approach:

1. LTA (Sigma Cat # L-2515 Lot 89F4061) was prepared to mg / ml in sterile endotoxin-free and pyrogen-free salt solution and stored at -70 ° C. A 750 µg / ml stock solution was prepared from this by dissolving 1.41 ml of LTA and 2.34 ml of brine. Final concentration of 300 μg / 400 μΐ per rat.

2. EB3-1 purified ascites (batch 6) were 4.5 mg / ml and were diluted in DPBS to a final concentration of 3.33 mg / ml. The dose administered per rat was 600 μΐ containing 2 mg (10 mg / Mg) sonicated for 1.5 min.

3. Nine three-month-old Lewis rats weighing about 195 grams were used. All were narcotized by inhalation of metaphane.

4. Rats were divided into three groups,

A. Control n = 1: They received 200 μΐ DPBS in both sides of the pleural cavity. They received 600 μΐ of sterile endotoxin-free and pyrogen-free saline, injected into the tail vein one hour after pleural infection.

B. LTA and EB3-1 treated: n = 4 Received

150 μΐ cavity, injected.

LTA at 200 μΐ

They got 600 μΐ in the tail

DPBS to both sides of the pleural (10 mg / 'Kg, 2 mg total) veins one hour after pleural

EB3-1

C. Only subject to LTA: n = 4 They received 150 μΐ LTA in 200 μΐ DPBS in both sides of the pleural cavity. get

600 μΐ DPBS injected into the tail vein one hour after pleural infection.

5. Rats are. gave the infections between ... the third .. and the fifth rib with the needle no. 30, with straight tip, and 1 cm ³ syringe. Tail vein infections were given with prior heating of rats under a heating lamp to make the veins more prominent, and were performed with needle number. 26 and 3 cm ³ syringe.

6. Rats were back in cages 3.5 hours after tail vein infection, 4.5 hours overall.

7. Rats were killed by metaphan inhalation.

8. The peritoneal cavity was opened to expose the peritoneum. A small incision was made on each side of the peritoneum and each side was washed with 1 ml of heparinized DPBS using a blunt tip of a 1 cm ³ syringe (without needle) and the liquids were combined.

9. Total volume was recorded, cells counted and centrifuged. They were suspended in 100 μΐ saline, and each suspension was coated onto a slide to stain with different fast dyes for differential analysis.

The results of this experiment are shown in FIG. 3, which shows that EB3-1 is effective in inhibiting LTA-induced pleuritis. The antibody inhibited PMN migration into the pleural cavity by 80%.

Example 6

This example demonstrates that the bacterial products Lipoplysaccharide (LPS) and Lipoteichoic acid (LTA) induce ELAM-1 expression on vascular endothelial cells. To achieve this, the ability of human neutrophils (PMN) to bind to cultured human endothelial cells was measured following prior in vitro treatment with various concentrations of LPS and LTA.

Approach

1. Stock solutions of LPS (Lot # 36F4019) and LTA (Sigma

Cat # L-2515 Lot 89F4061) were prepared for 1 mg / ml by sonication in saline just prior to use. Stock solutions were diluted with fresh EGM-UV medium. (Clonetics) for working concentrations of 100 µg / ml, 50 µg / ml, 25 µg / ml, 12 µg / ml, 6 µg / ml, 3 µg / ml, 1.5 µg / ml, 0.75 µg / ml,

0.37 μg / ml, 0.19 μg / ml and 0.09 μg / ml. One 96-well cluster dish] (Costar) was removed from the incubator following growth of HUVEC cord # 115 cells, passage 4, to confluence on gelatin. EGM-UV media or the same volume of LPS or LTA working dilutions, each dilution was analyzed in triplicate, and the plate was placed back in a 37 ° C incubator with 5% CO 2 for 4 hours.

3. Neutrophils (PMN) were prepared as in Example 1. PMNs were kept at room 1.04 x 10 ⁸ PMN and in some buffer but albumin.

Test plate temperature throughout the preparation. Yield resuspended at 6 x 10 ^5/25 μΐ of which containing 5 mg / ml of bovine serum stimulated HUVEC cells were removed from the incubator and the wells were washed twice with RPMI 1640 containing 5 mg / ml bovine serum albumin (BSA).

After the second wash, the wells were filled with 100 μΐ of the same buffer.

.

5. 0.025 ml of cell suspension was added to the wells of the stimulated assay plate.

6. Plates were incubated for 5 minutes at 37 ° C.

7. Unbound cells were removed from the wells of the assay plate by systematic resuspension using a p200 multichannel pipette followed by addition and withdrawal of 0.2 ml medium.

8. Remove all medium from the wells and add 50 μΐ of solubilization buffer. This consisted of citrate buffer (24.3 ml of 0.1 M citric acid, 10.5 g / 500 ml + 25.7 ml of 0.2 M sodium phosphate, 1.42 g / 500 ml and SQ H ₂ 0 to 100). ml) containing 0.1% NP-40 detergent.

9. Incubate the plate on a rotary shaker for 10 minutes and then add 0.05 ml OPDA (8 mg o-phenylenediamine, Sigma Cat. No. P-1526, 8 μΐ 30% H ₂ O ₂ and 10 ml _{) to each} well. citrate buffer (as above) was allowed to develop for 15 minutes and then 25 μΐ 4 MH ₂ SO ₄ was added ^to each well to stop the reaction.

10. Control reagent mix was prepared by mixing 100 μΐ volumes of solubilization buffer and OPDA solution and 50 μΐ 4 MH ₂ SO ₄ .

11. 100 μΐ of the supernatant was collected from every two wells and transferred to a flexible ELISA assay plate at 30 nm.

The plate was scanned at 492 (Falcon). minutes.

The results show that the neutrophils most effective in this experiment are demonstrated by both LPS and LTA induce on human endothelial between 0.2 and 5 µg / ml. The LTA was shown in FIG.

adhesion cells.

most efficient between

4, which LPS molecule was

5 and 50 μg / ml. At the optimal concentration of each compound, LPS induced neutrophil adhesion 2-3 times higher. as LTA.

Example 7

This example demonstrates that neutrophil adhesion to LPS and LTA-activated endothelium observed in Example 6 is mediated by ELAM-1.

The optimal time of endothelial activation by each endotoxin was also investigated. We measured the ability of human neutrophils (PMN) to bind to LTA and LPS activated cultured human endothelial cells at various times after addition of the activating agent, in the presence or absence of EB3-1.

Approach

1. Stock solutions of LPS (Lot # 36F4019) and LTA (Sigma

Cat # L-2515 Lot 89F4061) were prepared by sonication to 1 mg / ml in saline solution just prior to use. Stock solutions were diluted with fresh EGM-UV medium (Clonetics) to working concentrations of 10 µg / ml LTA and 0.2 µg / ml LPS. One 48-well cluster dish (Costar) was removed from the incubator following implanting HUVEC cord # 117 cells, passage 4, to confluence on gelatin. In all

2 hours, 8 hours, points, .. T hours, 6 hours, 8 hours, 18 hours. and 24 hours. medium .i. /.

The wells were removed with 0.5 ml of fresh EGM-UV LPS working dilutions.

Pasteur pipette medium (Clonetics) (three wells) or LTA into a 37 ° C incubator, 4 hrs, each containing (three wells), with 5% CO 2 for 4 hours.

3. Neutrophils (PMN) were prepared as in Example 1. PMNs were kept at room temperature throughout the preparation. Resuspended. to 2 x 10 ^ / 50 μΐ in the same buffer but containing 5 mg / ml bovine serum albumin.

4. Assay plate with stimulated HUVEC cells was removed from the incubator and the wells were washed twice with RPMI 1640 containing 5 mg / ml bovine serum albumin (BSA). After the second wash, the wells were filled with 300 μΐ of the same buffer. The medium was again removed from one well of each triad and replaced with 300 μΐ of hybridoma culture supernatant containing EB3-1 mAb. The plate was incubated for 10 minutes at 37 ° C and then removed from the incubator.

5. 0.025 ml of the PMN suspension was added to the wells of the stimulated test plate.

6. Plates were incubated for 5 minutes at 37 ° C.

7. Unbound cells were removed from the wells of the assay plate by systematic resuspension using a repeat dosing pipette followed by addition and withdrawal of 0.3 ml medium.

8. All media was removed from wells and added.

200 μΐ of solubilization buffer. This consisted of citrate buffer (24, 3 ml 0.1 M citric acid, 10.5 g / 500 ml +

25.7 ml of 0.2 M sodium phosphate dibasic, 1.42 g / 500 ml and SQ H ₂ to 100 ml) containing 0.1% NP-40 detergent.

9. The plate was incubated on a rotary shaker for 10 minutes and then 0.2 ml of OPDA solution (8 mg o-phenylenediamine, Sigma Cat # P-1526, 8 μΐ 30% H ₂ O ₂ and 10 ml) was added to each well. citrate buffer [as above]). The reaction was allowed to develop for 15 minutes and then 50 μΐ 4 MH ₂ SO ₄ was added to each well to stop the reaction.

10. Reagent Control Mixture ... was prepared by mixing 100 µL. volumes of solubilization buffer and OPDA solution and 50 μΐ 4 MH ₂ SO ₄ .

11. 100 μΐ of the supernatant was collected from every two wells and transferred to a flexible ELISA assay plate (Falcon). The plate was scanned spectrophotometrically at 492 nM within 30 minutes.

The results of this experiment are shown in FIG. 5, which shows that LPS and LTA induction of the neutrophil adhesion molecule on human endothelial cells has a peak between 4 and 6 hours. incubation with endothelial cells. This time course coincides with the pattern shown earlier for the induction of ELAM-1 (by) TNF and IL-β. At each time point tested, PMN adhesion can be completely inhibited by the anti-ELAM-1 mAb, indicating that both LPS and LTA induce ELAM-1 expression on human endothelium. ELAM-1 expression induced by these compounds decreases sharply after 6 hours of exposure, showing kinetics similar to that of IL-β activation.

Example 8

These examples demonstrate that the ability of EB3-1 to inhibit lethality induced by LPS is not dependent on complement fixation.

Experiments were previously performed to determine the relative blocking activity of EB3-1 F (ab) ' ₂ fragments and acid-treated EB3-1. First, it has been shown that the action of pH 2.5 on intact IgG ₃ permanently destroys complement fixation capabilities. (Winkelhake, J. et al., 1980, J. Biol. Chem. 2822-2828, incorporated herein by reference).

The adhesion of ⁵¹ Cr labeled HL-60 cells to IL-β activated endothelial cells was measured in the presence of titrated amounts of two preparations of EB3-1 mAb (acid treated Ig and F (ab ') ₂ ). 50% inhibition of binding was achieved with F (ab ') ₂ at ca.

0.5 μ9 / πι1. About 2 μ9 / πι1 Ig treated with acid produces equivalent adhesion inhibition.

We also iodinated (radioactive iodine) modified forms of EB3-1 and determined half-lives to:

two unmodified EB3-1 pH 2.5 treated with EB3-1 EB3-1 F (ab ') ₂ alpha-phase (min)

3.2

1.3 β-phase (min)

356

408

136

The EB3-1 blood purification profile is presented in FIG. 6. Based on these data and calculations of the area under the curve, we used two modified forms in the rat LPS-induced lethality model. The animals were dosed to give approximately the same peak concentrations and area under the curve for all three forms of EB3-1 between two and four hours after lethal LPS dosing. Like this: 4 groups of 10

iv lethal doses of LPS at time T = 0

EB3-1 forms as follows:

I - no therapy

EB3-1 - i.v. bolus 5 mg / kg at

III - EB3-1 acid treated - i.v.b.

IV - EB3-1 F (ab) ₂ - 7.5

5.0

2,5 received

GROUP

GROUP

II

GROUP

GROUP of animals received

T = 2 hours all mg / kg mg / kg mg / kg

T = 4 This dosing regimen approximates the same blood purification as at 5 mg / ml. Results, mechanism (s) of protection with complement fixation. potentially toxic human complement and / or referred to the F _c receptor.

is shown not

Plus, we are abilities

1.0 ml / kg

- 5 mg / kg hour hour hour area under the curve obtained with unmodified EB3-1 in FIG. 7, suggest that elaborately linked procedures to avoid mouse IgG3 fix cells by binding somehow showed this

While the present invention is described in some detail by way of illustration and examples in order to achieve clarity and clarity, it is clear that certain changes and modifications can be made within the scope of the appended claims.

Claims (8)

Pharmaceutical between cell-adherent adhesion carriers and the treatment of a disease involving mediated immunoglobulin, ELAMom-1, contains a pharmaceutical that recognizes a functional epitope on ELAMe-1 · Pharmaceutical for the treatment of a disease comprising ELISA-mediated medial cell adhesion according to item 1, wherein said immunoglobulin is 19G3. The pharmaceutical composition for treatment comprises treating intercellular adhesion-mediated disease ELAM-1 according to claim 1, characterized in that the immunoglobulin is secreted by the cell line A.T.C.C · Accession number HB 10591 4th Pharmaceutical composition for the treatment of a disease involving intercellular adhesion mediated ELAMom-1, characterized in that said immunoglobulin is incorporated into the liposome. Pharmaceutical for the treatment of a disease involving intercellular adhesion mediated ELAM-1 according to claim 4, characterized in that said liposome is an anti-inflammatory chemotherapeutic agent. Use of the pharmaceutical composition of step 1 for the manufacture of a medicament against a disease comprising intercellular adhesion mediated by ELAM-1, wherein said composition comprises an immunoglobulin which recognizes a functional epitope on ELAM-1. 7. The use of the pharmaceutical composition of item 1 for the manufacture of a medicament against a disease comprising ELAM-1 mediated intracellular adhesion, wherein said composition comprises an immunoglobulin and wherein said immunoglobulin is IgGs. 8- Use of a pharmaceutical composition according to item 1 for the manufacture of a medicament against a disease comprising ELAM-1 mediated intracellular adhesion, wherein said composition comprises an immunoglobulin and wherein said immunoglobulin is secreted by a cell line designated as A · T.C · C · accession number HB 10591. 9- Use of the pharmaceutical composition of item 1 for the manufacture of a medicament against a disease involving intercellular adhesion mediated by ELAM-1, wherein the disease involving intercellular adhesion is an inflammatory response. 10- Use of a pharmaceutical composition according to item 1 for the manufacture of a medicament against a disease involving intercellular adhesion mediated by ELAM-1, wherein the disorder comprising intercellular adhesion is septic soc- 11- Use of a pharmaceutical composition according to item i for the manufacture of a medicament against a disease involving intercellular adhesion mediated by ELAM-1 _? wherein the disease involving intercellular adhesion is adult respiratory distress syndrome. 12 · Use of the pharmaceutical composition of item 1 for the manufacture of a medicament against a disease involving intercellular adhesion mediated by ELAM-1? wherein the disease involving intercellular adhesion is sepsis, accompanying the injury. 13 · Use pharmaceutical composition according to point 1 for production 1 i e k u against a disease involving med z. i cellular ad héz iu ELAM-1 mediated, wherein said immunoglobulin is incorporated into the liposome · 14. Use of the pharmaceutical composition of item 1 for the manufacture of a medicament against a disease comprising ELAM-1 mediated intercellular adhesion. wherein said liposome encapsulates an anti-inflammatory chemotherapeutic agent.