DD297417A5 - INTERCELLULAR ADHAESION MOLECULAR-2 AND ITS BINDING LIGANDS - Google Patents

Abstract

The invention relates to the intercellular Adhaesionsmolekuel-2 * which is involved in the process by the lymphocytes to detect and migrate Entzuendungsherde, and to attach during the inflammation of cellular substrates. The invention relates to such molecules, sieve analyzes for the detection of such molecules and antibodies that are able to bind such molecules. The invention also includes application for adhesion molecules and for the antibodies that can bind them. {Intercellular adhesion molecule-2; Anti-ICAM-2 antibodies; Inflammation; recombinant DNA; pharmaceutical preparation}

Description

Text to pictures

Figuri: cell number cell number

relative fluorescence intensity-relative fluorescence intensity

% bound -% bound

control-control

FIG. 4: Arrangement of ICAM-2 and amino-terminal domains of ICAM-1

For this 4 pages drawings

Background of the invention

The invention relates to intercellular adhesion molecule-2 ("ICAM-2"), which participates in the process by which lymphocyte populations recognize and attach to cell substrates so that they can migrate to sites of inflammation and interact with cells during inflammatory reactions The invention further relates to ligand molecules capable of binding to the intercellular adhesion molecules ICAM-2 and to the field of application of the intercellular adhesion molecule and the ligand molecules.

Description of the essence of the invention

Leukocytes must be able to attach to cell substrates in order to protect the host cell against foreign invaders, e.g. B. bacteria or viruses, can adequately defend. Eisen, HW, (in "Microbiology", 3rd ed., Harper & Row, Philadelphia, PA [1980], pp. 290-295 and 381-418) gives an excellent review of the defense system, which must attach to endothelial cells In addition, they must attach to antigen-containing cells for normal, specific immune responses and then attach to appropriate target cells to allow the virus-infected or tumor cells to be disrupted Recently, leukocyte surface molecules involved in the mediation of such attachment processes have been identified using hybridoma technology In summary, it can be stated that monoclonal antibodies to human T cells (Davignon, D., et al., Proc. Natl. Acad. USA, 78, 4535-4539 [1981]) and mouse spleen cells (Springer, T., et al., Eur. J. Immunol., 9, 301-306 [1979]), which attached to leucocyte surfaces and inhibited the described attachment related functions (Springer, T., et al. a., Fed. Proc. 44, 2660-2663 [1985]). The molecules identified by these antibodies were termed Mac-1 and lymphocyte function-associated antigen-1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer found on most lymphocytes (Springer, T., et al., Immunol., Rev. 68: 11-115 [1982]). These two molecules, as well as a third molecule, ρ 150.95 (whose distribution in the tissue resembles that of Mac-1), play a role in cellular adhesion (Keizer, G., et al., Eur. J. Immunol., 15, 1142-1147 [1985]). It has been found that the described leukocyte molecules belong to a family of glycoproteins (Sanchez-Madrid, F., et al., J. Exper., Med., 158, 1785-1803 [1983]; Keizer, GD, et al., Eur. J. Immunol. 15, 1142-1147 [1985]), which are referred to as the CD-18 glycoprotein family. This glycoprotein family consists of heterodimers that have an alpha chain and a beta chain. Although the alpha chain was different for each antigen, it was found that the beta chain was largely retained (Sanchez-Madrid, F., et al., J. Exper. Med. 158, 1785-1803 [1983]). The beta chain of the glycoprotein family (sometimes referred to as "CD-18") has a molecular weight of 95 kd, while it has been determined that the molecular weight of the alpha chains ranges from 150 kd to 180 kd (Springer, T.A. , et al., Fed. Proc., 44, 2660-2663 [1985]). Although the alpha subunits of the membrane proteins do not have as much homology as the beta subunits, a close analysis of the alpha subunits of the glycoproteins revealed that they had considerable Similarities exist: Sanchez-Madrid, F. et al., (J. Exper. Med., 158, 586-602 [1983]); J. Exper. Med., 1785-1803 [1983]) provide an overview of the similarity of the alpha- and beta Subunits of the LFA-1 Related Glycoproteins Ef has been identified as a group of individuals unable to express normal levels of any member of this family of adhesion proteins on the cell surface of their leukocytes (Anderson, DC , etc., Fed. Proc. 44, 2671-2677 [1985]; Anderson, D. C, u. a., J. Infect. Dis. 152, 668-689 [1985]). Lymphocytes from these patients showed in vitro in vitro defects that antagonized in normal subjects whose CD-18 family of molecules by antibodies

had been observed defects resembled. In addition, because of the inability of their cells to attach to cell substrates, these individuals were incapable of normal immune response (Anderson, DC, et al., Fed. Proc., 44, 2671-2677 [1985]; Anderson, DC _1, et al. Infect. Dis., 152, 668-689 [1985]). These data indicate that immune responses are attenuated when lymphocytes are unable to attach normally due to the lack of functional adhesion molecules of the CD-18 family.

In summary, therefore, the ability of leukocytes to maintain an animal healthy and viable requires that they be able to attach to other cells (eg, endothelial cells). It has been found that this attachment requires contacts between cells involving specific receptor molecules. These receptors enable a leukocyte to attach to other leukocytes or to endothelial or other nonvascular tents. It has been observed that the receptor molecules of the cell surface are closely related. People whose leukocytes lack these cell surface receptor molecules suffer from chronic and recurrent infections as well as other clinical symptoms, e.g. B. disturbed antibody reactions.

Since leukocyte adhesion is involved in the process by which foreign tissue is identified and rejected, the understanding of this process in the fields of organ and tissue transplantation, allergy and oncology is of high value.

Summary of the invention The invention relates to the intercellular adhesion molecule-2 (ICAM-2) and its functional derivatives. The invention relates

also antibodies and antibody fragments capable of inhibiting the function of ICAM-2, as well as others

Inhibitors of the function of ICAM-2. The invention also includes the diagnostic and therapeutic use

all described molecules.

In particular, the invention encompasses the intercellular adhesion molecule ICAM-2 or a functional derivative of this molecule,

which is essentially free of natural contaminants.

The invention also relates to ICAM-2 containing at least one polypeptide from the group comprising:

(a) -S-S-F-G-Y-R-T-L-T-V-A-L-;

(b) -D-E-K-V-F-E-V-H-V-R-P-K-;

(c) -G-S-L-E-V-N-C-S-T-T-C-N-;

(d) -H-Y-L-V-S-N-I-S-H-T-D-V-;

(e) -S-M-N-S-N-V-S-V-Y-Q-P-P-;

(f) -F-T-I-E-C-R-V-P-T-V-E-P-;

(g) -G-N-E-T-L-H-Y-E-T-F-G-K-; (h) -T-A-T-F-N-S-T-A-D-R-E-D-; (I) -H-R-N-F-S-C-L-A-V-L-D-L-; (j) -M-V-H-V-T-V-V-S-V-L-L-;

(k) -S-L-F-V-T-S-V-L-L-C-F-I-; and (I) -M-G-T-Y-G-V-R-A-A-W-R-R-.

The invention also relates to a recombinant or synthetic DNA molecule capable of ICAM-2 or one of its

encode or express functional derivatives.

Moreover, the invention relates to an antibody and especially a monoclonal antibody capable of binding to a Molecule selected from ICAM-2 and a functional derivative of ICAM-2 group. The invention also relates to a hybridoma cell capable of producing the monoclonal antibody. The invention includes a method of producing a desired hybridoma cell that produces an antibody that is useful in

is capable of binding to ICAM-2 or its functional derivative, comprising the steps of:

(a) immunizing an animal with an immunogen selected from the group comprising: an ICAM-2 expressing cell, a membrane of an ICAM-2 expressing cell, ICAM-2, carrier-bound ICAM-2, a peptide fragment of ICAM-2 and a peptide fragment of ICAM-2 linked to a carrier;

(b) fusion of the spleen cells of the animal with a myeloma cell line;

(c) formation of antibody-secreting hybridoma cells by the fused spleen and myeloma cells and

(d) screening the hybridoma cells for the desired hybridoma cell capable of producing an antibody that binds to ICAM-2.

The invention also encompasses a method of treating inflammation resulting from the reaction of the specific defense system of a mammalian subject comprising administering to the subject in need of such treatment a sufficient amount of an anti-inflammatory agent to suppress the inflammation, the anti-inflammatory agent comprising the group comprising: an antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a Nir.ht immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 family of molecules.

The invention also includes a method for suppressing metastasis of a tumor cell line in the hematopoietic system, wherein the cell has a member of the CD-18 family (especially LFA-1) for migration; In this method, a patient requiring such treatment is administered an amount of an agent sufficient to suppress metastasis; wherein the agent is selected from the group comprising: an antibody capable of binding to ICAM-2; a toxin-derivatized antibody capable of binding to ICAM-2

tie; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 family of molecules.

The invention also includes a method for suppressing the growth of an ICAM-2 expressing tumor cell comprising administering to a patient in need of such treatment a sufficient amount of an agent to suppress growth, the agent being selected from the group comprising : an antibody capable of ICAM-? to bind; a toxin-derivatized antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 molecule family; a toxin-derivatized member of the CD-18 family of molecules; and a toxin-derivatized functional derivative of a member of the CD-18 family of molecules.

The invention also relates to a method for detecting the presence of an ICAM-2 expressing cell, the method comprising:

(a) incubating the cell or an extract of the cell in the presence of a molecule of nuolein, wherein the nucleic acid molecule is suitable for hybridization to ICAM-2 mRNA;

(b) determining whether the nucleic acid molecule has hybridized to a complementary nucleic acid molecule contained in the cell or in the extract of the cell.

The invention also relates to a pharmaceutical composition comprising:

(a) an anti-inflammatory agent selected from the group consisting of an antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2 and a Nicnt immunoglobulin antagonist other than ICAM-1 or a member of the CD-18 family of molecules, either alone or in combination with (b) an immunosuppressive agent.

Brief description of the figures

Figure 1 shows the binding of transfected COS cells expressing ICAM-1 and ICAM-2 to plastic material with LFA-1 coating. A) COS cells transfected with ICAM-1 cDNA were panned onto LFA-1 coated plates, and ICAM-1 expression was determined by indirect immunofluorescence flow cytometry with anti-ICAM-1 monoclonal antibody RR1 / 1 analyzed as primary MAb. Unscanned cells (dotted line), non-adherent cells (dashed line), adherent cells (solid line). B) ⁶¹ Cr-labeled transfected COS cells expressing ICAM-1 or ICAM-2 were ligated to LFA-1 coated plastic in the presence of MAb.

Figure 2 shows the nucleotide and amino acid sequence of ICAM-2. The amino acid sequence is numbered starting with the first residue after the predicted cleavage site of the signal peptide. The hydrophobic signal peptide and transmembrane sequences (TM) are underlined. Potential N-linked glycosylation sites are boxed. The putative polyadenylation signal AATACA! St is marked with a prime at the top. Both strands of ICAM-2 cDNA were amplified within CDM8 by sequential synthesis of the complementary oligonucleotide primers as well as termination sequencing of the dideoxynucleotide chain (Sanger, F., et al., Proc. Natl. Acad. See U.S.A. 74,5463-5467 [1977]). sequenced according to the recommendations of the manufacturer (Sequenase, US Biochemical).

Figure 3 shows the results of analyzes of RNA and DNA hybridization. Northern (A and B) and Southern (C) blots were hybridized to 1.1 kb ³² P-labeled ICAM-2 cDNA (A and C) and rehybridized to 3 kb ³² P-labeled ICAM-1 cDNA (B). (A and B) 6 μg poly (A) ⁺ RNA from the Burkitt lymphoma cell line, Ramos (lane 1), endothelial cells (lane 2), endothelial cells stimulated with LPS for 3 hours (lane 3), an EBV immobilized B lymphoblastoid cell line, BBN (lane 4), epithelial cell carcinoma cell line, HeLa (lane 5), T lymphoma cell line, Jurkat (lane 6) and SKW-3 (lane 7), and a promonocyte cell line, U 937 (lane 8). (C) 6 \ genomic DNA from B-cell lines BL-2 (lanes 1 and 4), ER-LCL (lanes 2 and 5) and Raji (lanes 3 and 6) were digested with EcoRI (lanes 1-3) or Hindill (tracks 4-6) open-minded. ICAM-2 and ICSM mKNAs are indicated by arrows.

Figure 4 shows the relationship between ICAM-2 and ICAM-1. The entire 201 residue extracellular sequence of ICAM-2 was probed using the ALIGN program (Dayhoff, M.O., et al., Methods Enzymol., 91, 524-545 [1983]) and by inspection of residues 1-185 of ICAM-1 assigned so that maximum match resulted. The ICAM-2 residues are numbered. Matching parts are boxed. D1 and D2 indicate the border of the Ig-like domain of ICAM-2 and ICAM-1. Predictions for the β-strand (Chou, P.Y., et al., Biochemistry 13, 211-245 (1974) of ICAM-2 are primed above, while those of ICAM-1 are underlined.

Description of the preferred application examples

One aspect of the present invention relates to the discovery of a natural ligand for binding to LFA-1. Molecules, such as B. belonging to the CD-18 family molecules involved in the process of cell adhesion are referred to as "adhesion molecules".

I. LFA-1 and ICAM-1

The leukocyte adhesion molecule LFA-1, upon immunity and inflammation, mediates a variety of interactions between lymphocytes, monocytes, natural killer cells, and granulocytes, on the one hand, and other cells, on the other hand (Springer, T.A., et al., Ann., Rev. Immunol, 5, 223-252 (1987)).

LFA-1 is a receptor for intercellular adhesion molecule-1 (ICAM-1). It is a surface molecule that is constitutively expressed on some gland tissues and induced on others in inflammation (Marlin, SD et al., Cell 51, 813-819 [19871, Dustin, ML et al., J. Immunol., 137, 245-254]. Dustin, ML et al., Immunol.Today 9,213-21511988]; U.S. Patent Application Serial Nos. 07 / 019,440, filed February 26, 1987, and U.S. Patent Application Serial No. 07 / 250,446, registered on February 28, 1987; September 1988, both applications incorporated herein for comparative purposes). LFA-1 functions in both antigen-specific and antigen-independent interactions between T cell toxins, T helper and natural killer cells, granulocytes and monocytes, and other cell types (Springer, TA et al., Ann. Rev. Immunol., 5,223-253 [1987]; Kishimoto, TK et al., Adv. Immunol. [1988, in press]). LFA-1 is a leukocyte integrin with noncovalently associated α and β glycoprotein subunits of 180 and 95kD.

ICAM-1 is a single-chain glycoprotein whose mass varies between 76-114kD, depending on the cell type. It belongs to the Ig extended family with five C-like domains (Dustin, ML et al., Immunol., Today 9,213-215 (1988), Staunton, DE et al., Cell 52,925-933 [1988]; Simmons, D. et al., Nature 331,624- 627 [1988]). ICAM-1 is associated with cytokines, wio e.g. IFN-γ, TNF and IL-1 are highly inducible on a variety of cell types (Dustin, M.L. et al., Immunol., Today 9, 213-215 [1988]). Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes (Dustin, ML et al., J. Immunol., 137, 245-254 (1986); Dustin, MLua, J. Cell. Biol Dustin, MLua, J.Exp.Med., 167, 1323-1340, 1988.) Adhesion is achieved by pretreatment of lymphocytes with LFA-1 MAb or pretreatment of the other cell with ICAM-1 MAb 137 (Dustin, M.L., et al., J. Immunol., 137, 245-254 [1986]; Dustin, ML et al., J. Cell Biol. 107, 323-331 [1988]; Dustin, ML et al., J. Exp. Med. 1323-1340 [1988]). Identical results with purified ICAM-1 in artificial membranes or in Petri dishes show that LFA-1 and ICAM-1 function as a receptor for each other (Marlin, SD et al., Cell 51, 813-819 [1987]; Makgoba, MW et al., Nature 331, 86-88 [1988]). For clarity, they are referred to herein as "receptor" or "ligand". Further descriptions of ICAM-1 can be found in US patent applications Ser. 07 / 045,963; 07 / 115.798; 07 / 155.943; 07 / 189,815 or 07 / 250,446. All applications in their entirety have been included herein for comparison purposes.

II. ICAM-2

A second LFA-1 ligand other than ICAM-1 has been postulated (Rothlein, R., et al., J. Immunol., 137, 1270-1274 (1986); Makgoba, MW et al., Eur. J. Immunol., 18, 637-640). 1988]; Dustin, ML et al., J. Cell Biol. 107, 323-331 [1988]). The present invention relates to this second ligand, referred to as ICAM-2 (short for "intercellular adhesion molecule-2"). ICAM-2 differs from ICAM-1 in its distribution in the cell and lack of cytokine induction. "ICAM-2 is an integral membrane protein with 2 Ig-like domains, while ICAM-1 has 5 Ig-like domains (Staunton, DE, et al., Cell 52,925-933); Simmons, D. et al., Nature 331, 624-627 [1988]) is the close relationship between ICAM-2 and two of the most N-terminal domains of ICAM-1 (34% identity), which is narrower than that between ICAM-1 or ICAM-2 and other members of the Ig extended family indicates a subset of Ig-like ligands that bind to the same integrin family-associated receptor.

III. cDNA cloning of ICAM-2

Any of a variety of methods can be used to clone the ICAM-2 gene. One method involves examining a shuttle vector library of cDNA inserts (derived from an ICAM-2 expressing cell) for the presence of an insert containing the ICAM-2 gene. Such analysis can be performed by transfecting cells with the vector and then assaying for expression of ICAM-2.

Preferably, ICAM-2 cDNA is identified by a novel modification of the method of Aruffo and Seed (Seed, B. et al., Proc Natl Acad., USA 84, 3665-3369 [1987]) to identify the ligands of adhesion molecules. In this method, a cDNA library is made from ICAM-2 expressing cells (eg, cell lines of endothelial cells or Ramos, BBN B lymphoblastoids, U937 monocytes, or SKW3 lymphoblastoids). The cDNA library is preferably made from endothelial cells. This bank is used to transfect cells that do not normally express ICAM-2 (eg, COS cells). The transfected cells are placed in a Petri dish previously provided with an LFA coating. COS cells transfected with either the coding sequences of ICAM-1 or ICAM-2 and expressing one of these ligands on their cell surfaces will anneal to the surface of the Petri dish on LFA-1. Unaffected cells are washed off and the attached cells are then removed from the Petri dish and grown. The recombinant ICAM-1 or ICAM-2 expressing sequence of these cells is then extracted and sequenced to determine whether it encodes ICAM-1 or ICAM-2.

In a preferred embodiment of the method described, an anti-ICAM-1 antibody is added to the Petri dish to prevent adhesion of cells expressing ICAM-1. The binding of ICAM-2 transfected COS cells to LFA-1 is inhibited by EDTA and an anti-LFA-1 monoclonal antibody (MAb), but not by an anti-LFA-1 monoclonal antibody. Thus, in this embodiment, ICAM-1 expressing cells are incapable of adhering to the Petri dish by ICAM-1, and most of them are therefore washed away along with any other non-adherent cells.

As a result, only ICAM-2 expressing cells are able to adhere to the petri dish. In this way, cDNA clones are screened for expression in COS cells as well as by panning on ligated COS cells using functionally-active, purified LFA-1 previously bound to plastic petri dishes. After scanning, the non-adherent cells have no ICAM-2 ⁺ cells, while the adherent cells released from the LFA-1 coated plastic shell by EDTA are almost exclusively ICAM-2 ⁺ . Adhesion of ICAM ⁺ -cellone to the LFA-1 coated plastic slides can be inhibited with RR1 / 1 anti-ICAM-MAb.

Thus, according to this method for cloning cDNA for ICAM-2, a cDNA library of endothelial cells is prepared which has both the ICAM-1-dependent and the ICAM-1-independent components of LFA-1-dependent adhesion (Dustin, M wherein a suitable Plasrnid, such as the plasmid vector CDM8 used. L. Others, J. cell. Biol. 107.321 to 331 (1988)), Transfected COS cells are incubated in LFA-1-coated petri dishes in the presence of anti-ICA M-1 MAb incubated to reduce the likelihood that ICAM-1 cDNAs will be isolated. Adherent cells are eluted with EDTA and the plasmids are isolated and amplified in E. coli. After about three transfection, adhesion and plasmid isolation cycles and size fractionation respectively, the plasmids can be analyzed by digestion with restriction endonuclease. Approximately Va of the plasmids having inserts greater than 1, okb showed adhesion to LFA-1 by transfection upon incorporation into COS cells.

Alternatively, a cDNA clone of ICAM-2 can be constructed using the genetic code (Watson, JD, in: "Molecular Biology of the Gene", 3rd ed., WABenjamin, Inc. Menlo Park, CA [1977], p .356-357) to determine the sequence of a polynucleotide capable of encoding the ICAM-2 protein.

A clone of the ICAM-2 cDNA can also be obtained by identifying the amino acid sequences of the peptide fragments of the iCAM-2 PfOtoin and then using the genetic code to construct oligonucleotide probe molecules capable of carrying the ICAM-2 cDNA. Encode peptide. The probes are then used to detect (by hybridization) those portions of a cDNA library (prepared from the cDNA of ICAM-2 expressing cells) encoding the ICAM-2 protein.

Such or similar techniques described make it possible to generate genes for human aldehyde dehydrogenases (Hsu, LC et al., Proc. Natl. Acad. Sci. USA 82, 3771-3775 [1985]), fibronectin (Suzuki, S. et al., Eur Biol. Organ J. 4,2519-2524 [19851], the human estrogen receptors (Walter, P., et al., Proc. Nt I. Acad., U.S.A., 82, 7889-7893 [1985]), the plasminogen activator of the tissue type (Pennica, D., et al., Nature 301, 214-221 [1983]) and the complementary DNA of the alkaline phosphatase, which arises in humans when reaching the Goburtstermins in the placenta (human term placental alkaline phe sphatase complementary DNA) (Kam, W et al., Proc. Natl. Acad., See. USA, 82, 8715-8719 [1985]). In another alternative method of cloning the ICAM-2 gene, a library of expression vectors is created by cloning DNA, or better cDNA, from a cell capable of expressing ICAM-2 in an expression vector. The library is then screened for members capable of expressing a protein that binds to the anti-ICAM-2 antibody and has a nucleotide sequence capable of encoding polypeptides having the same amino acid sequence as ICAM-2 or fragments of ICAM-2.

The cloned ICAM-2 gene obtained by using any of the described methods can be operatively linked to an expression vector and incorporated into bacteria or eukaryotic cells to produce ICAM-2. Methods for such manipulations are described by Maniatis, T. et al., Supra. and are well known to those skilled in the art.

IV. The Agents of the Present Invention: ICAM-2 and its Functional Derivatives, Agonists and Antagonists The invention relates to ICAM-2, its "functional derivatives" and its "agonists" and "antagonists".

A. Functional derivatives of ICAM-2

A "functional derivative" of ICAM-2 is a compound that has a biological (functional or structural) biological activity that is substantially similar to a biological activity of ICAM-2. The term "functional derivatives" includes the "fragments," "variants , "Analogs" or "chemical derivatives" of a molecule.

A "fragment" of a molecule, such as ICAM-2, refers to any polypeptide moiety of the molecule Fragments of ICAM-2 that exhibit! CAM-2 activity and are soluble (i.e., not membrane bound) are particularly preferred.

A "variant" of a molecule, such as ICAM-2, refers to a molecule that is substantially similar in structure or function to either the entire molecule or a fragment thereof.

An "analog" of a molecule, such as ICAM-2, refers to a molecule whose function is substantially similar to the entire molecule or a fragment thereof.

One molecule is "substantially similar" to another molecule if both molecules have substantially similar structures or similar biological activity, provided that two molecules have similar activity, they are considered variants, since this term is used herein even if the structure of one of the molecules does not recur in the other or if the sequence of amino acid residues is not identical.

As used herein, a molecule is referred to as a "chemical derivative" of another molecule if it contains additional chemical moieties that are not normally part of the molecule, such as the solubility, absorption, biological half-life, etc. of the molecule The proportions may either reduce the toxicity of the molecule, eliminate or reduce undesirable side effects of the molecule, etc. Components capable of mediating such effects are described in "Remington's Pharmaceutical Sciences" (1980).

"Toxin-derivatized" molecules are a special class of "chemical derivatives." A "toxin-derivatized" molecule is a molecule (such as ICAM-2 or an antibody) that contains a toxin moiety, the binding of such a molecule to the cell Any toxin moiety may be used, but preferably toxins per se, such as ricin toxin, holeratoxin, diphtheria toxin, radioisotopic toxins, membrane toxins, etc., are preferred. Channel-forming toxins, etc. Methods for coupling such moieties to a molecule are well known to those skilled in the art.

Functional derivatives of ICAM-2 having up to about 100 residues can be conveniently prepared by in vitro synthesis. If desired, such fragments may be modified by reacting targeted amino acid residues of the purified or crude protein with an organic derivatizing agent capable of reacting with selected side chains or terminal residues. The covalent derivatives thus obtained can be used to identify residues that are important for biological activity.

Cystelnyl residues are usually treated with α-haloacetates (and corresponding amines), ζ.B. Chloroacetic acid or Chloroacetamide, reacted to obtain carboxy methyl or carboxyamidomothyl derivatives. Cysteinyl residues are also

by reaction with bromotrifluoroacetone, α-bromo-β- (6-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulphide, methyl-2-pyridyl disulphide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole derivatized.

Histidyl residues are derivatized by reaction with diethylprocarbonate at a pH of 5.5-7.0 because of this agent

acts on the histidyl side chain relatively specific. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

Terminal lysinyl and amino residues are reacted with succinic or carboxylic anhydrides. Derivatization with

These agents cause reversal of the charge of the lysinyl residues. Other suitable agents for derivatizing amino-containing residues include imidoesters, e.g. methyl picolinimidate; Pyridoxal phosphate, pyridoxal;

chloroborohydride; trinitrobenzene; O-menthyl-isourea; 2,4-pentanedione and the transaminase-catalyzed Reaction with glyoxylate. Arginyl residues are prepared by reaction with one or more conventional reagents, i.a. a. phenylglyoxal; 2,3 Butanedione; 1,2-cyclohexanedione and ninhydrin, modified. The derivatization of arginine residues is due to the high pK,

the functional guanidine group required the reaction under alkaline conditions. Besides, these can

Reactants react with the lysine groups as well as with the epsilon-amino group of arginine. The specific modification of the tyrosyl residues per se has been extensively studied, with the incorporation of ν -jaral markers into Tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane base .. β attention

was given. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are used to prepare labeled proteins for use in radioimmunoassay

Method iodinated with ¹²⁵ I or ¹³¹ I, the chloramine-T method is particularly suitable. Carboxyl side groups (aspartyl or glutamyl) are prepared by reaction with carbodiimides (R'-N-C-N-FO, e.g.

1-Cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3 (4-azonia-4,4-dimethylpentyl) carbodiimide, selectively modified.) In addition, aspartyl and glutamyl residues are converted to asparaginyl by reaction with ammonium ions. and

Converted to glutaminyl residues. Derivatization with bifunctional agents is indicated for the cross-linking of a functional derivative molecule of ICAM-2

a water-insoluble carrier matrix or surface used in the method of cleaving a fusion polypeptide of a functional derivative of ICAM-2 to release and recover the cleaved polypeptide. Frequently used crosslinking agents include e.g. 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde,

N-hydroxysuccinimide ester, e.g. Esters with 4-azldosalicylic acid, homobifunctional imidoesters, including Disuccinimidester, such as. B. S ^ '- Dithiobislsuccinimidylpropionat), and bifunctional maleimides, z. Bis-N-maleimido-1,8-octane. Derivatizing agents, e.g. Methyl 3 - [(p-azidophenyl) dithio] propioimidate gives photoactivatable intermediates,

which are able to form crosslinks in the presence of light. Alternatively, reaction-friendly, water-insoluble matrices, e.g. Cyanogen bromide activated hydrocarbons and those disclosed in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440 to reactive substrates described for

Protein immobilization used. Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues. When Alternatively, these residues are deamidated under mildly acidic conditions. Each of the two forms of these radicals is in Scope of the invention included. Further modifications include the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of Seryl or theyl residues, the methylation of the a-amino groups of lysine, arginine and histidine side chains (T.E.

"Proteins: Structure and Molecule Properties", W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]), the acetylation of the

N-terminal amine and in some cases the amidation of the C-terminal carboxyl groups. Functional derivatives of ICAM-2 with altered amino acid sequences may also be due to mutations in the DNA

getting produced. The nucleotide sequence encoding the ICAM-2 gene is shown in FIG. Such variants slept e.g.

Deletions of residues within the amino acid sequence shown in Figure 2 or insertions or substitutions. each Combination of deletion, insertion and substitution can also be made to the final construct

provided it has the desired activity. It goes without saying that the mutations made in the DNA encoding the variant should not force the sequence out of reading frame and preferably should not create any complementary regions which could give rise to a secondary mRNA structure (see EP publication

Patent Application No. 75444). At the genetic level, these functional derivatives are normally derived by site-directed mutagenesis Nucleotides in the DNA encoding the ICAM-2 producing the DNA encoding the functional derivative, and

obtained by subsequent expression of the DNA in the Rokombina'.ionszellkultur. The functional derivatives normally have the same qualitative biological activity as the naturally occurring analogue. With regard to the

However, they can be considerably different from those of the normally produced ICAM-2 molecule

differ.

Although the site is predetermined for the incorporation of an amino acid sequence variation, this is needed in the mutation as such

not to be the case. So z. For example, to optimize the performance of a mutation at a particular location, random mutagenesis may be performed on the target codon or region, and the expressed functional derivatives of ICAM-2 may be tested for the optimal combination of desired activity. Methods for substitution mutations at predetermined locations in the DNA with a known sequence are well known, such as. As the site-specific mutagenesis.

Ci Preparation of a molecule of a functional derivative of ICAM-2 according to the invention is preferably carried out by site-specific mutagenesis of the DNA which encodes a previously prepared functional derivative or a non-variant version of the protein. Site-directed mutagenesis allows the production of functional derivatives of ICAM-2 by use of specific ollgonucleotide sequences encoding the DNA sequence of the desired mutation as well as a sufficient number of contiguous nucleotides to obtain a primer sequence of sufficient size and sequence complexity to form a stable duplex DNA on both sides to deliver the deletion site to be bridged. Normally, a primer of about 20 to 25 nucleotides in length with about 6 to 10 residues on either side of the sequence site to be altered is used. In general, the method of site-directed mutagenesis is well known in the art, for which publications such as Adelman et al. a., DNA 2,183 (1983), an example whose disclosure is included herein for comparison.

As can be seen, the site-directed mutagenesis method normally utilizes a phage vector that occurs in both single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors, such as e.g. As the M13 phage of brass u. al., "Third Cleveland Symposium on Macromolecules and Recombinant DNA," Editors A.Walton, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein for comparative purposes. "" This phage is readily available for purchase and its use is of the person skilled in the art Alternatively, plasmid vectors containing a replication starting point in the form of a single-stranded phage (Veira et al., Moth. Enzymol., 153, 3 [1987]) can be used to obtain single-stranded DNA.

In general, site-directed mutagenesis according to the invention is carried out by first obtaining a single-stranded vector which contains within its sequence a DNA sequence encoding the corresponding protein. An oligonucleotide primer carrying the desired mutated sequence is prepared, which is generally synthetic by the method of Crea et al. a., Proc. Natl. Acad. Sei (USA) 75, 5765 (1978) happens. This primer is then adhered to the single-stranded protein sequence-containing vector and exposed to DNA polymerizing enzymes, such as. As the polymerase-I Klenow fragment of E. coil to complete the synthesis of the mutation-bearing strand. This catfish is given a mutated sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform suitable cells, e.g. JM101 cells are used, and clones are selected which contain the recombinant vectors carrying the mutated sequence.

After selection of such a clone, the mutated protein region can be harvested and inserted into a vector suitable for protein production. In general, it is an expression vector of a type that can be used to transform a suitable host cell.

Amino acid sequence deletions generally range from about 1 to 30 residues, preferably 1 to 10 residues, and are normally adjacent to one another. Deletions may also include an immunoglobulin domain, e.g. domains 1 or 2 of ICAM-2.

Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions from one residue to polypeptides of indefinite length, as well as insertions of one or more amino acid residues within a sequence. Intrasequence insertions (i.e., insertions within the complete sequence of the ICAM-2 molecule) may generally be from about 1 to 10 residues, preferably 1 to 5 residues. An example of a terminal insertion involves fusion of a signal sequence, whether heterologous or homologous to the host cell, with the N-terminus of the molecule to allow for the secretion of the functional derivative of ICAM-2 by the recombinant host cells facilitate. The third group of functional derivatives are those in which at least one amino acid residue in the ICAM-2 molecule - and preferably only one - has been removed and another residue has been inserted in its place. Such substitutions are preferably made according to the following table, if the fine tuning of the features of the ICAM-2 molecule is desired.

Table 1

Onginalrest Typical substitutions Ala gly; ser bad lys Asn gin; his Asp glu Cys ser Gin asn Glu asp Gly ala; pro His asn; gin Ue leu; val Leu ile; val Lys arg; gin; glu mead leu; tyr; ile Phe met; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

Significant changes in functional or immunological identity are made by selecting substitutions that are more extensive than those listed in Table 1, d. H. by selecting residues whose action is different from maintaining (a) the structure of the polypeptide-cell pellet in the substitution area, for example as a layer or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the mass of the side-chain. The generally expected substitutions are those in which (a) glycine and / or proline is substituted, deleted or inserted by another amino acid; (b) a hydrophilic residue, e.g. Seryl or threonyl, for (or through) a hydrophobic residue, e.g. Leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (c) one cystaine residue is substituted for (or by) another residue; (d) a moiety having an electropositive side chain, e.g. Lysyl, arginyl or histidyl for (or by) a moiety having electronegative charge, e.g. Glutamyl or aspartyl, is substituted; or (e) a moiety having a bulky side chain, e.g. Phenylalanine, is substituted for (or by) a group which has no such side chain, e.g. Glycine.

It is not expected that the majority of deletions and insertions, and in particular substitutions, will cause profound changes in the characteristics of the ICAM-2 molecule. However, if it is difficult to predict in advance the exact effect of substitution, deletion or insertion, it will be appreciated by those skilled in the art that the effect will be determined by routine screening assays. Thus, a functional derivative is usually obtained by site-directed mutagenesis of the nucleic acid encoding the native ICAM-2 molecule, expression of the altered nucleic acid in a recombinant cell culture, and optional purification from the cell culture, e.g. By immunoaffinity adsorption to a column containing an antibody to anti-ICAM-2 molecule (to absorb the functional derivative by binding at least one of the remaining immune epitope).

Mutations designed to increase the affinity of ICAM-2 can be directed by the insertion of the amino acid residues present in the same positions in ICAM-1. Ana'og to such a mutant ICAM-2 molecules can be prepared, which have no N-linked CHO at the same positions in ICAM-1.

The activity of the cell lysate or purified functional derivative of the ICAM-1 molecule is then tested for the desired trait in a suitable screening assay. Thus, for example, a change in the immunological character of the functional derivative, e.g. For example, the affinity for a particular antibody is measured by a competitive immunoassay. Changes in immunomodulatory activity are measured by the appropriate test. Modifications of such protein properties as redox or thermal stability, biological half-life, hydrophobicity, susceptibility to proteolytic degradation, or carrier-to-multimeric tendency are evaluated by methods well known to those of ordinary skill in the art.

B. agonists and antagonists of ICAM-2

An "agonist" of ICAM-2 is a compound that enhances the ability of ICAM-2 to perform any of its biological functions An example of such an agonist is an agent that enhances the ability of ICAM-2 to bind to a human cellular receptor or to bind a viral protein.

An "antagonist" of ICAM-2 is a compound that reduces the ability of ICAM-2 or prevents it from fulfilling any of its biological functions. Examples of such antagonists include ICAM-1, functional derivatives of ICAM-1 , Anti-ICAM-2 antibodies, anti-LFA-1 antibodies, etc.

Cell aggregation assays described in U.S. Patent Application Serial Nos. 07 / 045,983; 07 / 115.798; 07 / 155.943; No. 07 / 189,815 and 07 / 250,446, respectively, the applications of which are collectively incorporated herein for purposes of comparison, are capable of measuring LFA-1 dependent aggregation and may be used to determine the extent of the agents affect the aggregation triggered by ICAM-2 / LFA-1. Such assays can be used to identify agonists and antagonists of ICAM-2. Antagonists may act by interfering with the aggregating mediating ability of LFA-1 or ICAM-2. In addition, non-immunoglobulin (i.e., chemical) agents can be assayed using the assay described to determine whether they affect the aggregation of ICAM-2 / LFA-1 as agonists or antagonists.

C. Anti-ICAM-2 antibodies

The preferred immunoglobulin antagonist of the present invention is an antibody to ICAM-2. Suitable antibodies can be obtained in various ways.

An antigen molecule, such as. ICAM-2, is expressed naturally on the surface of lymphocytes. Thus, introduction of such cells into a suitable animal by intraperitoneal injection, etc., will result in the production of antibodies capable of binding ICAM-2 or members of the CD-18 family of molecules. If desired, the serum of such an animal may be removed and used as a source of polygonal antibodies capable of binding these molecules.

Alternatively, anti-ICAM-2 antibodies can be obtained by adapting the method of Seiden, R.F. (Publication of European Patent Application No. 289034) or Seiden, R.F. (Science 236, 714-718 (1987)) According to such an adaptation of this method, the cells of a suitable animal (ie, a mouse, etc.) are transfected with a vector capable of either the intact ICAM-2 The production of ICAM-2 in the transfected cells of the animal will elicit an immune response in the animal and cause the animal to produce anti-ICAM-2 antibodies.

Alternatively, anti-ICAM-2 antibodies can be obtained by introducing ICAM-2 or its peptide fragments into a suitable animal. The immunized animal will produce polygonal antibodies in response. The use of peptide fragments of ICAM-2 makes it possible to obtain region-specific antibodies that react only with the epitope (epitopes) contained in the peptide fragments used to immunize the animals. However, it is preferable to remove spleen cells from the animals (which have been immunized as described above), to fuse these spleen cells to a myeloma cell line, and to allow the formation of hybridoma cells by the fusion cells. These hybridoma cells secrete monoclonal antibodies capable of binding to ICAM-2.

The on the besch; The way hybridoma cells are obtained can be tested by various methods

to detect those hybridoma cells secreting an antibody capable of binding to ICAM-2. In a preferred screening assay, such molecules are identified by the ability to inhibit the aggregation of ICAM-2 expressing ICAM-1 non-expressing cells. Antibodies capable of inhibiting such aggregation are then further tested to determine whether they support aggregation by binding to ICAM-2 or to an antibody

Inhibit members of the CD-18 molecule family. In such a screening test, any agent that is suitable may be ICAM-2 from the CD-18-Molecule family to be used. So z. B. bound by the antibody Antigen z. Example by immunoprecipitation and polyacrylamide gel electrophoresis. Fs is possible between Antibodies that bind to members of the CD-18 family of molecules and antibodies that bind to cells, the LFA-1,

but do not express ICAM-2 (or vice versa).

The ability of an antibody to bind to a cell expressing LFA-1 but not ICAM-2 can be assessed by methods

which are generally used by persons with average professional knowledge. These include immunoassays (especially those that use immunofluorescence), cell agglutination,

Filter binding studies, antibody precipitation, etc. In addition to the described functional derivatives of ICAM-2, other agents corresponding to the present invention include Invention can be used for the treatment of viral infections or inflammation, antibodies against ICAM-2,

anti-idiotypic antibodies against anti-ICAM-2 antibodies and receptor molecules or fragments of such molecules capable of binding to ICAM-2.

The antibodies to ICAM-2 (or functional derivatives of ICAM-2) of interest are those of use

it is either polyclonal or monoclonal antibody.

The anti-idiotypic antibodies of interest to the invention are capable of competing with (or under Exclusion of) ICAM-2 binding. Such antibodies can, for. B. be obtained by an antibody to a Cultured anti-ICAM-2 antibody and then the antibody is tested for the ability to adapt to the natural Bind binding ligands of ICAM-2. Since the molecules of the CD-18 family are capable of binding to ICAM-2, the administration of such molecules (e.g. Heterodimers having both alpha and beta subunits, or as molecules containing fragments of one or more

containing both subunits) compete with HRV for binding to ICAM-21 present on the cells (or exclude HRV).

The anti-aggregation antibodies of the present invention can be identified and titrated in various ways. So z. For example, the ability of the antibodies to differentially bind to ICAM-2 expressing cells (such as activated Endothelial cells) and their inability to bind to cells that do not express ICAM-2. Suitable tests

the cell aggregates are those described in US patent applications Ser. 07 / 045,963; 07 / 115.798; 07 / 155,943; 07/1, £ 9,815 and 07 / 250,446, respectively. The applications in their entirety are included in the present application for purposes of comparison. Alternatively, the ability of the antibodies to bind to ICAM-2 or peptide fragment of ICAM-2 can be measured. Those of ordinary skill in the art will readily recognize that the above assays can be modified or reordered to yield a variety of potential screening assays that are suitable for antibodies capable of binding to ICAM -1 to distinguish from or identify those that bind to the CD-18 family of molecules.

In a preferred method, the antibody may be selected for its ability to bind to COS cells which ICAM-2 express but not bind to COS cells that do not express ICAM-2. D. Preparation of the Agents of the Invention

The agents of the invention may be induced by natural processes (such as by causing an animal, a plant, fungi, bacteria, etc., to produce a non-immunoglobulin antagonist of ICAM-2, or by inducing an animal to produce polygonal antibodies capable of binding to ICAM-2); by synthetic methods (such as by using the Merrifield method for synthesizing polypeptides used to synthesize ICAM-2, functional derivatives of ICAM-2 or protein antagonists of ICAM-2 (either immunoglobulin or non-immunoglobulin) by hybridoma technique (such as the production of monoclonal antibodies capable of binding to ICAM-2) or by the recombinant technique (such as the production of agents of the invention in various host cells [ie yeasts, Bacteria, fungi, mammalian cultured cells, etc.] or from recombinant plasmids or viral vectors.) The choice of which method to use will depend on such factors as convenience, desired yield, etc. It is not necessary to produce one use only one (s) of the described methods, methods or techniques for the anti-inflammatory agent; Methods, methods and techniques may be combined to obtain a particular agent.

V. Uses of ICAM-2 and its functional derivatives, agonists and antagonists A. Suppression of inflammation One aspect of the invention is based on the ability of ICAM-2 and its functional derivatives, with receptors of the CD-18 Family of molecules, in particular with LFA-1, or with virus proteins (such as, for example, the proteins of the rhinovirus, etc.) in Interacting. Its ability to interact with members of the CD-18 glycoprotein family

ICAM-2 can be used to suppress (i.e., prevent or attenuate) inflammation.

The term "inflammation" as used herein includes both the reactions of the specific defense system

also the reactions of the nonspecific defense system.

As used herein, the term "specific defense system" refers to the component of the immune system that is based on the Presence of specific antigens reacts. An inflammation is due to a response of the specific defense system

attributed when the inflammation is caused or mediated by or related to a response of the specific defense system. Examples of inflammation that respond to the specific defense system

is due to shoot the response to antigens such as rubella virus, autoimmune diseases, T cells-mediated delayed hypersensitivity response (observed, for example, in subjects who are "positive" in Mantoux-Tesi), etc. Chronic inflammatory diseases and rejection of transplanted tissues and organs are further examples inflammatory reactions of the specific defense system.

As used herein, a response of the "unspecific defense system" refers to a response mediated by leukocytes that lack immunological memory, such as granulocytes and macrophages. As used herein, inflammation is the result of a response of the patient nonspecific defense system when the inflammation is caused or mediated by or is related to a nonspecific defense system response Examples of inflammation that are due, at least in part, to a response of the nonspecific defense system include inflammation associated with the following conditions associated with: adult respiratory distress syndrome (ARDS) or septicemia or trauma syndrome of multiple organ damage; damage to the myocardium or other tissues following repair of a circulatory disorder; acute glomerulonephritis; secondary arthritis; dermatoses with acute inflammatory conditions n components; acute purulent meningitis or other inflammatory disorders of the central nervous system; Heat damage; hemodialysis; Leukapheresis: ulcerative colitis; Crohn's disease; necrotizing enterocolitis; Syndromes associated with granulocyte transfusion and cytokine-induced toxicity.

As already stated, the binding of the ICAM-2 molecules to the members of the CD-18 family of molecules is of eminent importance for cell adhesion. Through the adhesion process, lymphocytes are able to constantly monitor an animal for the presence of foreign antigens. However, although these processes are usually desirable, they also cause rejection of transplanted organs, rejection of tissue transplants, and numerous autoimmune diseases. Thus, any method of attenuating or inhibiting cell adhesion would be highly desirable in those receiving organ transplants (particularly kidney transplants), tissue transplants, and patients suffering from autoimmune diseases.

CD-18 family monoclonal antibodies inhibit numerous adhesion-dependent functions of leukocytes, including their binding to the endothelium (Haskard, D., et al., J. Immunol., 137, 2901-2906 (1986)), homotypic adhesions (US Pat. Rothlein, et al., J. Exp. Med. 163: 1132-1149 [1986]), antigen and mitogen-induced proliferation of lymphocytes (Davignon, D., et al., Proc. Natl. Acad. Sei., USA, 4535-4539 [1981]), the antibody formation (Fischer, A., et al., J. Immunol., 136, 3198-3203 [1986]) and the effector functions of all leukocytes, such as the lytic activity of the cytotoxic T cells (Krensky, AM, et al., J. Immunol., 132, 2180-2182 [1984]), macrophages (Strassman, G., et al., J. Immunol., 136, 438-4333 [1986]) and all cells involved in antibody-dependent cellular cytotoxic reactions (Kohl, S., et al., J. Immunol., 133, 2972-2978 [1984]). In all of these functions, the antibodies inhibit the ability of leukocytes to harden on the appropriate cell substrate, which in turn affects the end result. As far as these functions are concerned with the interactions between ICAM-2 and LFA-1, they can be suppressed with an anti-ICAM-2 antibody. Thus, monoclonal antibodies capable of binding to ICAM-2 can be used as anti-inflammatory agents in a mammal. Importantly, such agents differ from common anti-inflammatory agents in that they are capable of selectively inhibiting adhesion, and that they do not have other side effects or do not deleteriously affect conventional agents.

Because ICAM-2 is particularly soluble in its ability to act as an antibody against members of the CD-18 family, it can be used to suppress inflammation. In addition, the functional derivatives and antagonists of ICAM-2 can also be used to suppress inflammation.

1. Suppressor of delayed-type hypersensitivity reactions

In part, ICAM-2 molecules mediate adhesion events that are required to elicit inflammatory responses, such as: B. Delayed type hypersensitivity reactions. Thus, antibodies (especially monoclonal antibodies) capable of binding to ICAM-2 molecules have a therapeutic potential for mitigating or preventing such reactions.

Since ICAM-2 is an antagonist of the interaction between ICAM-1 and LFA-1, as an alternative ICAM-2 (particularly in solubilized form) or its functional derivatives can be used to suppress delayed-type hypersensitivity reactions.

These therapeutic applications can be used in different ways. First, a patient with delayed-type hypersensitivity reactions may be administered a monoclonal antibody-containing composition. Thus, such compositions could, for. To a person infected with antigens, e.g. Poisonum (Rhus toxicodendron), American lacquerum (Rhus vernix) etc. had contact. In the second embodiment, the monoclonal antibody capable of binding to ICAM-2 is administered to a patient together with an antigen to prevent a later inflammatory reaction. Thus, the additional administration of an antigen together with an ICAM-2 binding monoclonal antibody may cause a subject to temporarily tolerate the subsequent administration of this antigen.

2. Treatment of chronic inflammatory diseases

Since LAD patients who do not have LFA-1 do not produce an inflammatory response, it is believed that the antagonist of the natural ligand of LFA-1 - the ICAM-2 will also inhibit an inflammatory response. The ability of antibodies to ICAM-2 to inhibit inflammation provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases, such as: Lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes, Reynaud's syndrome, rheumatoid arthritis, etc. Such antibodies can also be used to treat psoriasis. In general, monoclonal antibodies capable of binding to ICAM-2 can be used to treat those diseases currently treatable by steroid therapy

 According to the invention, such inflammatory and immunological rejection reactions can be suppressed (i.e.

prevented or attenuated) by administering to a subject in need of such treatment an anti-inflammatory agent In amounts sufficient to suppress inflammation. Suitable anti-inflammatory agents include: an antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a non-immunoglobulin antagonist of ICAM-2 that is not LFA-1. Particularly preferred are anti-inflammatory agents consisting of a soluble functional derivative of ICAM-2. Anti-inflammatory treatment may also include the additional administration of an agent selected from the group consisting of: an antibody capable of binding to LFA-1; a functional derivative of an antibody, wherein said functional derivative is capable of binding to LFA-1; and a non-immunoglobulin antagonist of LFA-1.

The invention further includes the methods described for suppressing an inflammatory response of the specific ablation system in which the subject is additionally administered an immunosuppressant. Such an immunosuppressant is preferably administered at a lower dose (ie, a "less than optimal" dose) than would normally be required, and the use of a suboptimal dose is possible because of the synergistic action of the agents of the present invention Examples of suitable immunosuppressants include dexamethasone. Azathioprine, ICAM-1, cyclosporin A and so on.

3. Therapy of non-specific inflammation

The invention is based in part on the discovery that granulocyte-endothelial cell adhesion is due to the interaction of glycoproteins of the CD-18 family with the endothelium. Because cell adhesion is required for leukocytes to migrate to sites of inflammation and / or to perform various inflammatory function functions, agents that inhibit cell adhesion will attenuate or prevent inflammation. Such inflammatory responses are due to responses of the "non-specific defense system" mediated by leukocytes that lack immunological memory Granulocytes and macrophages are such cells As the term is used herein, inflammation is the result of a nonspecific immune system response, when the inflammation is caused or mediated by, or is related to, a response of the nonspecific system Examples of inflammation, which are, at least in part, due to a response of the nonspecific defense system, include inflammation associated with the following conditions: respiratory distress syndrome in adulthood (ARDS); a syndrome of multiple organ damage following septicemia or trauma; damage to the myocardium or other tissues following repair of circulatory disorders; acute glomerulonephritis; secondary arthritis; dermatoses with acute inflammatory components; a purulent meningitis or other inflammatory disorders of the central nervous system; Heat damage; Hemodialysis, leukapheresis; Ulcerative colitis; Crohn's disease; necrotizing enterocolitis; Syndromes associated with granulocyte transfusion and cytokine-induced toxicity.

The anti-inflammatory agents of the invention are compounds capable of specifically counteracting the interaction of the CD-18 complex on granulocytes with endothelial cells. Such antagonists include: ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 family of molecules.

B. suppressors of organ and tissue rejection

Because ICAM-2, especially in its soluble form, is able to act in the same way as an antibody against members of the CD-18 family, it can be used to suppress organ or tissue rejection caused by any of the cell adhesion-dependent functions is caused. In addition, anti-ICAM-2 antibodies and the functional derivatives and antagonists of ICAM-2 can also be used to suppress this rejection reaction. ICAM-2 and antibodies capable of binding to ICAM-2 can be used to prevent organ or tissue rejection or to modify autoimmune responses in mammals without the risk of side effects. Importantly, the use of monoclonal antibodies capable of detecting ICAM-2 may allow organ transplants to be made even between individuals whose HLA does not match.

C. Additive to antigenic material administered for therapeutic and diagnostic purposes. Immune responses to therapeutic or diagnostic agents, such as: B. bovine insulin, interferon, plasminogen factor (tissue activator) or mouse monoclonal antibodies severely limit the therapeutic or diagnostic value of such agents and may even cause diseases such. As the serum sickness cause. This situation can be remedied by using the antibodies of the invention. In this embodiment, such antibodies would be administered in combination with the therapeutic or diagnostic agent. The addition of antibodies prevents the recipient from recognizing the agent and thus prevents the recipient from giving an immune response. The absence of such an immune response causes the therapeutic or diagnostic agent to continue to be administered to the patient.

ICAM-2 (especially in solubilized form) or its functional derivatives are interchangeable in the treatment of diseases with ICAM-1 or antibodies capable of binding to LFA-1. Thus, such molecules can be used in solubilized form to inhibit rejection of organs or grafts. ICAM-2 or its functional derivatives can be used in the same way as anti-ICAM-2 antibodies to reduce the immunogenicity of the therapeutic or diagnostic agents.

D. suppressors of tumor metastasis

The agents of the invention may also be used to suppress the metastasis of a tumor cell of the hematopoietic system which requires migration of a functional member of the CD-18 family. According to this embodiment of the invention, a patient in need of such treatment receives an agent (e.g., an antibody capable of binding to ICAM-2; a toxin-derivatized antibody capable of acquiring ICAM-5 !, a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2;

functional derivative of! CAM-2; and a non-immunoglobulin antagonist of ICAM-2 which is not ICAM-1) in an amount necessary to suppress metastasis.

The invention also relates to a method of suppressing the growth of an ICAM-2 expressing tumor cell which comprises administering to a patient in need of such treatment a sufficient amount of an agent to suppress growth. Suitable agents include an antibody that is capable of binding to ICAM-2; a toxin-derivatized antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1; a toxin-derivatized member of the CD-18 family of molecules and a toxin-derivatized functional derivative of a member of the CD-18 family of molecules.

The invention also relates to a method for suppressing the growth of an LFA-1 expressing tumor cell which comprises administering a growth suppressing amount of a toxin to a patient in need of such treatment. Suitable toxins include a toxin-derivatized ICAM-2 or a toxin-derivatized functional derivative of ICAM-2.

E. suppressors of viral infections

Recently, the important group of rhinoviruses has been shown to challenge the position of ICAM-1 as a receptor (Greve, JM, et al., Cell 56, 839-847 [1989]; Staunton, DE, et al., Cell 66, 849-853 [1989 Tomassini, JE, et al., Proc Natl. Acad. See, USA 86,4907-4911 [1989], all of which are incorporated herein for comparative purposes). Rhinoviruses, members of Picornavirone's small, RNA-containing protein-encapsulated family, cause 40-50% of all common colds (Riieckert, RR, in: Field's Virology, Fields, BN, et al. (Ed.) Raven Press, NY [1985] p Sperber, SJ, et al., Antimicr. Agents Chemo., 32, 409-419 [1988], all of these references are included herein for comparative purposes.) Over 100 no immunological cross-reactivity rhinoviruses have been defined, 90% of which bind to ICAM -1.

Recently, it has been found that in addition to ICAM-1, the cell adhesion molecule CD4 and the complement receptor CR2 are challenged by HIV and EBV viruses, respectively, as virus receptors (Maddon, PJ, Cell 47, 333-348 [1986]; Fingeroth, JD, et al. Proc. Natl. Acad. Sci. USA 81, 4510-4514 [1984], these references are incorporated herein for comparative purposes). In addition, a molecule having an ICAM-1-like Ig domain structure which can be effective in tent adhesion acts as a receptor f, V the poliovirus. (Mendelsohn, CL, et al., Cell 56: 855-865 [1089]).

ICAM-2 and its functional derivatives can act as receptors for the attachment of viruses (especially lower serotype rhinoviruses) or infection. Thus, antibodies to ICAM-2 (or its fragments), ICAM-2 or functional derivatives of ICAM-2 can be used to block attachment or infection and thereby suppress viral infection.

F. Diagnostic and prognostic indications

Monoclonal antibodies capable of binding to ICAM-2 can be used to test for a patient. To calibrate sites where ICAM-2 expression and inflammation occurs. For this purpose, the monoclonal antibodies are labeled by use of radioisotopes, affinity tags (e.g., biotin, avidin, etc.), fluorescent labels, paramagnetic atoms, labeled anti-ICAM-2 antibodies, etc. for detection purposes. Those skilled in the art are well aware of methods for such labeling. A review of the use of antibodies in the clinic for diagnostic imaging has been reviewed by Grossman, H. B., Urol. Clin. North Amer. 13,465-474 (198o), Unger, E. C, u. a., Invest. Radiol. 20,693-700 (1985) and Khaw, B.A., et al., Science 209,295-297 (1980).

The presence of ICAM-2 expression can be improved by the use of binding ligands, such as. MRNA, cDNA or DNA which bind to ICAM-2 gene sequences or ICAM-2 mRNA sequences from ICAM-2 expressing cells. Methods for performing such hybridization assays are described by Maniatis, T., et al. in "Molecular Cloning, a Laboratory Manual", Coldspring Harbor, NY (1982) and by Haymus, BD, et al., "Nucleic Acid Hybridization, a Practical Approach", IRL Press, Washington, DC (1985), the references of which are included herein for comparative purposes.

The detection of foci of such antibodies for detection purposes indicates a location where expression of ICAM-2 or tumor development occurs. In one embodiment, this assay is performed for expression by taking tissue or blood samples and incubating these samples in the presence of antibodies that can be labeled. In a preferred embodiment, this is done in a non-bleeding manner by magnetic imaging, fluorography, etc. Such a diagnostic test can be used to monitor organ transplant recipients for signs of possible tissue rejection. Such assays may also be performed to assess a person's susceptibility to rheumatoid arthritis or other chronic inflammatory diseases.

For example, by radioactively labeling the antibody or antibody fragments, it is possible to detect the antigen by using radioimmunoassays A good description of a radioimmunoassay (RIA) is given in the book "Laboratory Techniques and Biochemistry in Molecular Biology" by Work TS, et al., North Holland Publishing Company, NY (1978), specifically in the chapter entitled "Introduction to Radioimmunoassay and Related Techniques" by Chard, T., which is hereby incorporated herein by reference is included. As an alternative, fluorescence, enzyme or other suitable markers may be used.

Examples of types of labels that may be used in the invention include, but are not limited to, enzyme labels, radioisotopic labels, nonradioactive isotope labels, fluorescent labels, toxin markers and chemiluminescent labels.

Examples of suitable enzyme markers include: platelet dehydrogenase, staphylococcal nuclease, della-5-steroid isomerase, yeast a cohold dehydrogenase, alpha glycerol phosphate dehydrogenase, triosephosphate isomerase, povidoxidase, alkaline phosphatase, asparaginase, glucose oxydase, beta-galactosidase, ribcuclease, urease, catalase, glucose-6 phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc.

Examples of suitable radioactive labels include ³ H, ¹¹¹ In, " ⁶ I," ¹¹ , ³² P, ³⁶ S, ¹⁴ C, ⁶¹ Cr, ⁶⁷ To, ⁶⁸ Co, ⁶⁹ Fe, ⁷⁶ Se, ¹⁶² Eu, ⁸⁰ Y , ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁸ Pd, etc. examples of suitable non-radioactive label include ¹⁶⁷ GD, ⁶⁶ Mn, ¹⁸² Dy, ⁶² Tr, ⁶⁶ Fe, etc. examples of suitable fluorescent markers include ¹⁶² Eu-markers, a fluorescein marker, a rhodamine marker, a phycoerythrin marker. a phycocyanin marker, an allophycocyanin marker, an o-pthaldehyde marker, a fluoresceamine marker, etc.

Examples of the chemiluminescent markers include a luminal marker, an isoluminal marker, an aromatic acridine ester marker, an imidazole marker, an acridine salt marker, an oxalate ester marker, a luciferin marker, a luciferase marker, an aequorin marker, etc.

Vl. Administration of the compositions of the invention

The therapeutic effects of ICAM-2 can be achieved by administering to a patient the entire ICAM-2 molecule or any therapeutically active peptide fragments thereof. Of particular interest are therapeutically active peptide fragments of ICAM-2 which are soluble.

ICAM-2 and its functional derivatives can be obtained either synthetically by the use of recombinant DNA technology or by proteolysis or a combination of these methods. The therapeutic value of ICAM-2 can be increased by the use of functional derivatives of ICAM-2 having additional amino acid residues used to enhance coupling to the carrier or to increase the activity of ICAM-2. The scope of the invention also includes functional derivatives prior to ICAM-2 in which certain amino acid residues are absent or contain altered amino acid residues as long as these derivatives have or affect a biological or pharmacological activity of ICAM-2.

Both the antibodies of the invention and the ICAM-2 molecule disclosed herein are "substantially free of natural contaminants" when preparations containing them are substantially free of materials with which these products are normally found and in the juvenile state ,

The invention extends to antibodies and their biologically active fragments (whether polygonal or monoclonal antibodies) which are capable of binding to ICAM-2. Such antibodies can be produced either by an animal, a tissue culture or by recombinant DNA technology.

In the administration of antibodies or their fragments capable of binding to ICAM-2, to a patient or to the administration of ICAM-2 (or a fragment, variant or derivative thereof) to a patient the dosage of the administered agent will depend on such factors as age, weight, height, sex, general condition, history, etc. In general, it is desirable to administer to the recipient an antibody dose in the range of about 1 pg / kg to 10pg / kg (patient body weight), although a lower or higher dose is also possible. When administering ICAM-2 molecules or their functional derivatives to a patient, it is desirable to administer the molecules in a dosage that is also from 1 pg / kg to 10pg / kg (body weight of the patient), although lower or higher Dosage is also possible. As will be noted, the therapeutically effective dose can be reduced if the anti-ICAM-2 antibody is additionally administered with an anti-LFA-1 antibody. As the term is used herein, a compound is considered to be administered in addition to a second compound if the administration of the two compounds occurred at such a short interval that both compounds can be detected simultaneously in the patient's serum.

Both the antibody capable of binding to ICAM-2 and auc. ICAM-2 itself can be administered to the patient intravenously, intramuscularly, subcutaneously, enterally or parenterally. When the antibody is administered to the ICAM-2 by injection, it may be by continuous infusion or bolus or multiple boi. The agents of the invention are to be administered to the recipients in an amount sufficient to suppress inflammation. An amount is considered sufficient for "suppressing" inflammation if the dosage, route of administration, etc. of the agent is sufficient to attenuate or prevent the inflammation. Anti-ICAM-2 antibodies or their fragments may be used either alone or in combination with one or more of the agents The administration of such a compound (s) may either serve a "prophylactic" or "therapeutic" purpose, and if administered prophylactically, dia immunosuppressive compound (s) prior to the occurrence of an inflammatory reaction or symptom (e.g., before, at, or shortly after) the time of organ or tissue transplantation, but prior to the onset of any of the organ rejection symptoms.) Prophylactic administration of Compound (s) serves to provide a subsequent inflammatory response (eg, the rejection of a transplant organ or tissue, etc.) to prevent or mitigate. When used therapeutically, the immunosuppressive compound (s) will be administered at (or shortly after) the onset of a symptom of actual inflammation (eg, organ or tissue rejection). Therapeutic administration of the compound (s) serves to attenuate actual inflammation (such as rejection of a transplanted organ or tissue). The anti-inflammatory agents of the present invention can thus be administered either before the onset of inflammation (to suppress an expected inflammation) or after the inflammation has occurred. Collaboration is considered "pharmacologically acceptable" when tolerated by a patient to whom it is administered, such an agent being considered to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence causes a demonstrable change in the physiology of the recipient.

The antibody of the invention and the ICAM-2 molecules can be prepared according to known methods for the preparation of pharmaceutically active compositions, whereby these materials or their functional derivatives are admixed to a pharmaceutically acceptable carrier. Suitable excipients and their preparation, including those for other human proteins, e.g. B. human serum albumin, suitable, z. Osol, A. (ed.), Mack, Easton PA [1980]), etc. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions contain effective Amount of anti-ICAM-2 antibody or ICAM-2 molecule or their functional derivatives together with a suitable amount of vehicle.

Other pharmaceutical methods can be used to control the duration of action. Controlled release preparations can be achieved by using polymers to complex with or absorb the anti-ICAM-2 antibodies or ICAM-2 or their functional derivatives. Controlled release can be achieved by selection of appropriate macromolecules (eg, polyester, polyamino acid, polyvinyl, pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate) and by concentration of the macromolecules and controlled release incorporation methods. Another possible method of controlling the duration of action of controlled release preparations is to use anti-ICAM-2 antibodies or ICAM-2 molecules or their functional derivatives in particles of a polymeric material, e.g. For example, polyester, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymer can incorporate. Alternatively, instead of incorporating these agents into polymer particles, it is possible to include these materials in microcapsules that are e.g. By coacervation techniques or by interfacial polymerization, e.g. As hydroxymethylcellulose or gelatin microcapsules or poly (methyl methacrylate) microcapsules or in colloidal drug release systems, such as. Liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules or in macroemulsions. Such methods are disclosed in "Remington's Pharmaceutical Sciences" (1980).

The invention further relates to a pharmaceutical composition comprising: (a) an anti-inflammatory agent (such as an antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is expressed in FIG capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; and a non-immunoglobulin antagonist of ICAM-2 which is not ICAM-1 and (b) at least one immunosuppressive agent suitable immunosuppressants are dexamethesone, azathioprine and cyclosporin A.

Having generally stated the nature of the invention, the understanding of the invention will be further appreciated by reference to the following examples, given by way of illustration, but not for the purpose of limiting the invention, unless expressly noted.

example 1 Cloning of ICAM-? -u) NA

For cloning cDNA capable of encoding ICAM-2, a modification of the method of Aruffo and Seed (Seed, B., et al., Proc. Natl. Acad., USA 84,3365-3369 (1987 ]) were used to select cDNAs by expression in COS cells to scan for ligand-bearing COS cells on functionally-active purified LFA-1 bound to plastic petri plates.

Specifically, LFA-1 was purified by immunoaffinity chromatography on TS 2/4 LFA-1 MAb Sepharose from the SKW-3 lysate and eluted at pH 11.5 in the presence of ₂ mM MgCl ₂ and 1% octylglucoside. LFA-1 (10pg / 200μΙ / cm3 plate) was bound to bacteriological petri dishes by diluting octylglucoside to 0.1% in PBS with 2nM MgCl ₂ and incubating overnight at 4 ° C. The plates were blocked with 1% BSA and stored in PBS / ₂ mM MgCl ₂ / 0.2% BSA / 0.025% azide / 5C Mg / ml gentamycin.

The synthesis of a cDNA library from LPS-stimulated umbilical vein endothelial cells according to the method developed by Gubler and Hoffmann was carried out as described by Staunton et al. (Staunton, DE, et al., Cell 52,925-933 (1988)) Stranges, the cDNA was ligated to Bst XI adapters (Seed, B., et al., Proc. Natl. Acad., Sci. USA 84,3365-3369 (1987)) and cDNAs> 600bp were amplified by low melting agarose gel electrophoresis (LMP). The cDNA was then ligated to CDM8 (Seed, B., Nature 329, 840-842 [1987]), incorporated into E. coli host MC1061 / P3 and plated to give 5x10 ⁵ colonies LB medium was suspended, pooled, and the plasmid was recovered by the standard alkali lysis method (Maniatis, T., et al., In "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory (1982).) Ten 10-cm plates COS cells grown to 50% were incubated with 10 μg / plate of the plasmid cDNA library transfected using DEAE-dextran (Kingston, RE, in Current Protocols in Molecular Biology, 9.0.1-9.9.6, Greene Publishing Associates [1987]). ICAM-2 is trypsin-resistant on endothelial and SKW-3 cells. COS cells were suspended three days after transfection by treatment with 0.025% trypsin / 1mM EDTA / HBSS (Gibco) and scanned on LFA-1 coated plates (Seed, B., et al., Proc. Natl. Acad. See, USA 84,3365-3369 [1987]), as described below for the ⁶¹ Cr-labeled COS cells. Adherent cells were detached by addition of EDTA up to 10 mM.

The plasmid was recovered from the adherent population of COS cells in Hirtscher's supernatant supernatants (Hirt, BJ, J. Mol. Biol., 26, 365-369 (1967).) E. coli strain MC1061 / P3 was then probed with the The plasmid was transformed, the colonies on the plates were suspended in LB medium, pooled, and the plasmid was prepared by the alkali-lysis method, and two more cycles of selection of LFA-1-adhered transfected COS cells and cycles for plasmid recovery were performed The pooled colonies obtained after the third cycle were grown to 100 ml LB medium with IB 1 g / ml tetracycline and 20 μg / ml ampicillin to saturate The plasmid was prepared and fractionated by 1% LMP agarose gel electrophoresis, and MC 1061 / P3 was transformed separately with plasmid of nine different size classes: individual plasmids from the fraction which had the highest activity in promoting the adhesion of the COS cells to LFA-1 The size of the insert was examined by digestion with Xb & l and tested in a COS cell adhesion test. He gave a plasmid with an ICAM-2 cDNA insert of 1.1 kb, pCDIC 2.27. Adhesion tests were carried out using the ICAM-2 plasmid pCDIC2, 27 or an ICAM-1 construct containing the 1.8kb Sail, KpnI fragment (Staunton, DE, et al., Cell 52, 925-933 [1988]) in CDM8 (2 μg / ml). 10cm plate) incorporated into COS cells using DEAE-Dextran. The COS cells were suspended three days after transfection with 0.025% trypsin / 1 mM EDTA / HBSS and labeled with ⁶¹ Cr. About 2 × 10 ⁵⁶¹ Cr-labeled COS cells in 2 ml PBS / 5% FCS / 2 mM MgCl ₂ / 0.025% azide (buffer) with δμρ / σιΙ of the MAb mentioned were grown in 6 cm LFA-1 coated plates for 1 hour incubated at 25 ° C. Non-adherent cells were removed by gentle shaking and three rinses with the buffer. Adherent cells were eluted by addition of EDTA to 1OmM and counted in the gamma counter.

The feasibility of this procedure was demonstrated using COS cells transfected with the previously cloned ICAM-1 cDNA (Figure 1a). ICAM-1 was expressed on 25% of the transfected cells. After palpation, the non-adherent cells had no ICAM-1 ⁺ cells, while the adherent cells detached with EDTA from the LFA-1 coated plastic were almost completely ICAM-I ⁺ . Adhesion of ICAM-1 ^ cells to LFA-1

coated plastic was inhibited with RR1 / 1ICAM-1 MAb. LFA-1 coatings on petri dishes were stable for> 5 cycles of COS cell adhesion and elution with EDTA. The plates were stored between runs in Mg ²⁺ at 4 ° C. For cloning of ICAM-2, a cDNA library of endothelial cells having both ICAM-1 dependent and ICAM-1 independent components of LFA-1-dependent adhesion was prepared in the plasmid vector CDM8 (Dustin, M. L, et al., J. Cell Biol., 107, 323-331 [1988]). Transfected COS cells were incubated in LFA-1 coated Petri dishes in the presence of ICAM-1 MAb to prevent isolation of ICAM-1 cDNAs. Adherent cells were eluted with EDTA and plasmids were isolated and amplified in E. coli. After three cycles of transfection, adhesion and plasmid isolation cycles and size fractionation, 30 plasmids were analyzed by restriction endonuclease digestion. Of three plasmids containing inserts> 1, Okb, a piasmide incorporated into COS cells by transfection showed adhesion to LFA-1. '

The isolated piasmid transferred the adhesion to LFA-1 to a greater percentage of the transfected cells. The percentage was approximately equal to that achieved in ICAM-1 transfection (Figure 1B). However, adhesion was not blocked by LFA-1 MAb (Figure 1B), unlike the ICAM-1 transfected cells by ICAM-1 MAb (Figure 1B). In addition, cells transfected with this piasmide did not react with a group of four ICAM-1 MAbs. Thus, all functional criteria for a cDNA encoding a second LFA-1 ligand were met and the ligand was designated "ICAM-2".

Example 2 Characterization of the ICAM-2 cDNA sequence

The 1052bp ICAM-2 cDNA sequence (Figure 2) contains an untranslated region at 62 bp 5 'and 167 bp 3'. An AATACA polyadenylation signal at position 1019, which in contrast to AATAAA occurs in about 2% of the vertebrate mRNA (Wickens, M., et al., Science 226, 1045-1051 (1984)), is followed at 1058bp by a poly (A) tail The longest open reading frame starts with the first ATG at position 63 and ends with a TAG termination codon at position 885. Based on the hydrophobicity analysis (Kyle, J., et al., J. Mol. Biol. 157, 105-132 (1982)) and the Use of amino acids at cleavage sites (Heijne, G. Nucleic Acids Research 14, 4883-4690 [1986]) predicted a 21 residue signal peptide (Figure 2) .The predicted mature sequence contains a putative extracellular domain from amino acid 1 to 201 followed by a 26-residue hydrophobic putative transmembrane domain and a 26-residue cytoplasmic domain Four loops of a presumed transmembrane segment with alpha helix are amphipathic, with treonine and serine residues falling to one side, suggesting the possibility of suggesting self-association or association with other membrane proteins at the membrane level. The cytoplasmic domain is unusually basic and, unlike most cytoplasmic domains, which are hydrophilic, have an average hydrophobicity. The predicted mass of the mature polypeptide is 28176 daltons which, if the six predicted N-linked glycosylation sites are utilized, would yield an ICAM-2 glycoprotein of about 46Kd.

Example 3 Analyzes of hybridization of DNA and RNA The isolated ICAM-2 cDNA clones were analyzed by both Northern and Southern hybridization. Both Northern blots were 6 μg of poly (A) ⁺ RNA which had been treated with a 1% agarose formaldehyde gel (Maniatis, T., et al. In: "Molecular Cloning:" A Laboratory Manual ", Cold Spring Harbor Laboratory (1982)), electrophoresed and separated on a Nylon membrane was electrically transferred (Zeta Probe, BioRad) used. The completion of the transfer was by UV transillumination of the gel and fluorescence photography of the blot confirmed. The genomic DNAs were washed five times with the manufacturer's recommended amount of EcoR I and Hind III endonucleases (New England Biolabs) open-minded. After electrophoresis through a 0.8% agarose gel, the DNAs were assayed for zeta

transfer. RNA and DNA blots were prehybridized and hybridized according to standard procedures (Maniatis, T., et al. In "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory [1982]) using ICAM-2 or ICAM-1 cDNAs generated by random priming (Boehringer Mannheim with a [ ^{32p] d} XTPs) were marked.

The 1.1 kb ICAM-2 cDNA hybridizes to a 1.4 kb poly (A) ⁺ mRNA and weakly to a 3 kb mRNA (Figure 3 A), which differs from the

3.3 kb and 2.4 kb ICAM-1 mRNA distinguishes (Fig.3B). mRNA was studied in cells classified as functional according to their function

Cells that have ICAM-1-dependent and second-ligand-dependent binding to LFA-1. ICAM-1 mRNA is strongly induced by LPS in endothelial cells (Figure 3B, lanes 2 and 3). In contrast, ICAM-2 mRNA

is strongly basally expressed in endothelial cells and not further induced by LPS (Figure 3A, lanes 2 and 3). This finding is consistent with the strong basal and non-inducible expression of the LFA-1 dependent, ICAM-1 independent

Metabolism in endothelial cells and the inducibility of the ICAM-1-dependent metabolic pathway (Dustin, M.L., et al., J.

Cell. Bio. 107, 323-331 [1988]).

ICAM-2 mRNA is present in a variety of cell types, among others. a. Ramos and BBN B lymphoblastoid, U 937 monocyte and SKWS lymphoblatoid cell lines (Fig. 3A, lanes 1,4,6 and 8) as seen from moderate or long autoradiography. It

SKW3, U937 and BBN of these cells were demonstrated to be LFA-1 dependent, ICAM-1 independent adhesion

LFA-1 ⁺ cells (Rothlein, et al., J. Immunol., 137, 1270-1274 [1986]; Makgoba, MW, et al., Eur. J. Immunol., 18, 637-640 [1988])

even after prolonged autoradiography shows no ICAM-2 mRNA (FIG. 3A, lane 5). The cell distribution of ICAM-2 is thus consistent with the ICAM-1-independent component of LFA-1-dependent adhesion.

Southern blots of genomic DNA (Figure 3D) hybridized with the ICAM-2 cDNA showed a single

predominant EcoR I fragment of 8.2 kb and a Hind III fragment of 14 kb, indicating a single gene in 8 kb, containing the majority of the coding information.

Example 4 Comparison of the amino acid sequences of ICAM-1 and ICAM-2 The amino acid sequences of ICAM-2 and ICAM-1 were compared because of their functional similarity as LFA-1 ligands. ICAM-1 belongs to the Ig extended family, and its extracellular domain consists entirely of five C-like domains. The 201 ICAM-2 extracellular domain comprising amino acids consists of 2 Ig C-like domains, with the suspected

Within the domain contained disulfide-bound cysteines 43 and 56 Rt ste are located apart from each other, and a predicted ß-Strengthen structure (Figure 4) is included. It is noteworthy that the two Ig-like domains of ICAM-2 have a 34% identity with the two most N-terminal ig-like domains of ICAM-1 in their amino acid sequence (Figure 4); Score 15 sd is above the mean, and has a 27% identity with the ICAM-1 domains 3 unr, 4, where the ALIGN score is 3 s.d. above the mean. Research in the NBRF and SWISS-PROT databases revealed homology to only partial domains when compared to other members of the Ig extended family, primarily HLA class II antigens. ICAM-2 has less-conserved residues characteristic of the IG domain than ICAM-1. ICAM 2 has 17% or 19% identity with the two N-terminal domains of the adhesion molecules NCAM (Cunningham, B. Α, et al., Science 236, 799-806 [1987]) and MAG (Saizer, J. L, et al., J. Cell. Bio). 104, 957-965 (1987)), while ICAM-1 has 19% and 20% identity, respectively. Lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 ilCAM-1) were identified by selecting MAb that blocked T lymphocyte-mediated killing or homotypic adhesion (Rothlein, R., et al., J. Med. Immunol., 137, 1270-1274 (1986); Davignon, D., et al., Proc. Nati, Acad., USA 78, 4535-4539 (1981)). In contrast, ICAM-2 was synthesized using a selection procedure for functional cDNA which does not require prior identification of the protein by biochemical or immunological methods Isolation of a cDNA for ICAM-2 confirms the postulated existence of a second LFA-1 ligand Distribution of mRNA for ICAM-2 on a limited number of cells , who are known for their ICAM-1-dependent and ICAM-1-independent adhesion to LFA-1, suggests that ICAM-2 might explain all observed ICAM-1-independent LFA-1-dependent adhesion.

ICAM-2 and the two N-terminal domains of ICAM-1 are much more similar than other members of the Ig extended family, indicating a subfamily of Ig-like molecules that bind to LFA-1. Importantly, the LFA-1 binding region of ICAM-1 was mapped by domain deletion and systematic amino acid substitution as domains 1 and 2. Consequently, there is both structural and functional homology. ICAM-2 is the second example of a member of the Ginger Ig family that binds to an integrin. Although there is little hierarchy among cell adhesion receptors, it has been demonstrated that in the integrins a number of extracellular matrix component receptors recognize multiple ligands (Hynes, RO, Cell 48, 559-554 (1987), Ruoslahti, E., et al., Science 238, 491) -497 (1978)).

Neither ICAM-1 nor ICAM-2 contains an RGD sequence, and thus the manner of recognition by LFA-1 may be different than that of the integrins that bind extracellular matrix components (Hynes, RO, Cell 48: 549-554 (1987); Ruoslahti, E., et al., Science 238, 491-497 (1987)). The cellular ligands recognized by Mac-1 and ρ 150.95 - leukocyte integrins closely related to LFA-1 - may belong to the same Ig Subfamily Recently, ICAM-1 has been shown to be a receptor for the important group of rhinoviruses that cause 50% of common colds, and ICAM-2 can also act as a receptor for rhinoviruses or other picornaviruses. ie, alleviating the attenuation) of infections caused by such viruses.

A family of ligands for LFA-1 underscores the importance of this recognition pathway and may be a mechanism for mediating the specificity and functional diversity. Some differences between ICAM-1 and ICAM-2 are of potential importance. ICAM-1 is inducible on most cells, while ICAM-2 expression is unaffected by cytokines on previously tested cells. It is expected that because of the three additional domains on ICAM-1, its LFA-1 binding site will be further away from the cell surface than ICAM-2, suggesting that in the interaction of LFA and ICAM-1. 2 requires closer contact between the cells than in the interaction between LFA-1 and ICAM-1. As the shear force of rinsing is increased, COS cells transfected with ICAM-2 are more easily detached than ICAM-1 transfected COS cells from LFA-1 coated plastic. This may be due to the smaller size of ICAM-2, making it less accessible on the artificial substrate for LFA-1. It may also be due to sequence differences that mediate affinity differences. The individual cytoplasmic domains of ICAM-1 and ICAM-2 may mediate different signals or cause a different localization on the cell surface. Depending on whether ICAM-1 or ICAM-2 is bound, the signaling or interaction of LFA-1 with the cytoskeleton may also be different. ICAM-1 and a second LFA-1 counter receptor, ICAM-2, thus represent a subfamily of the immunoglobulin (Ig) family (Staunton, DE, et al., Cell 52,925-933 (1988).) This reference is for reference herein was added). ICAM-1 has five Ig-like C domains, while ICAM-2 has two, which are particularly closely related to the amino-terminal domains of ICAM-1. ICAM-1 and ICAM-2, which are expressed on a variety of cell types, support various leukocyte adhesion-dependent functions, i.a. a. the induction and effector functions in the immune response. Expression of ICAM-1 can be strongly induced by cytokines, and thus the LFA-1 / ICAM-1 adhesion system can direct the migration and localization of leukocytes upon inflammation (Rothlein, R., J. Immunol., 137, 1270- 1274 (1986)); Marlin, SD, et al., Cell 51, 813-819 [19871, Kishimoto, TK, et al., Adv. Immunol., 46, 149-182 [1989]; Dustin, ML, et al., Immunol., Today 9, 213-215 [1988 ], all of these references are incorporated herein for brevity). ICMA-1 residues previously found to be important for LFA-1 binding are conserved in other ICAMs (Staunton, DE, et al., Nature 339, 61-64 (1989) for comparative purposes). Human ICAM-1 has 50% identity with mouse ICAM-1 and 35% identity with human ICAM-2 (Staunton, D.E., et al., Nature 339, 61-64 [1989]). The most important residues for LFA-1 binding - E34 and Q73 - are contained in both mouse ICAM-1 and human ICAM-2. This is consistent with the ability observed in both mouse ICAM-1 and human ICAM-2 (Staunton, D.E., et al., Nature 339, 61-64 [1989]) to bind to LFA-1. A D2 N-linked glycosylation site at N156, which affects LFA-1 binding, is also found in ICAM-2. Several residues important for rhinovirus-14 binding, i. Q58, G46, D71, K77 and R166 are not found in mouse ICAM-1 or in human ICAM-2 (Staunton, DE, et al., Cell 56, 849-853 [1989]) (this reference is incorporated herein for comparative purposes), which is consistent with the apparent inability of mouse cells (Colonno, RJ, et al., J. Virul., 57-12 [1986]) and ICAM-2 to bind rhinovirus-14.

While the invention has been described in conjunction with specific embodiments of the invention, it is to be understood that other modifications of the invention are possible, and this application includes any modification, use, or adaptation of the invention that substantially conforms to the principles of the invention, including those Variations from the present disclosure, which are part of the known or customary practice of the art to which the invention relates, or to the essential features are set forth, which are set forth in the following claims.

Claims (20)

1. ICAM-2 or a functional derivative, characterized in that it is substantially free of natural impurities. 2. iCAM-2 according to claim 1, characterized in that ICAM-2 is additionally capable of binding to a molecule present on the surface of a cell or to a virus. 3. ICAM-2 molecule according to claim 2, characterized in that the molecule contains at least one polypeptide from the group comprising: (a) -S-S-F-G-Y-R-T-L-T-V-A-L-; (b) -D-E-K-V-F-E-V-H-V-R-P-K-; (c) -G-S-L-E-V-N-C-S-T-T-C-N-; (d) -H-Y-L-V-S-N-I-S-H-T-D-V-; (e) -S-M-N-S-N-V-S-V-Y-Q-P-P-; (f) -F-T-I-E-C-R-V-P-T-V-E-P-; (g) -G-N-E-T-L-H-Y-E-T-F-G-K-; (h) -T-A-T-F-N-S-T-A-D-R-E-D-; (i) -H-R-N-F-S-C-L-A-V-L-D-L-; (j) -M-V-I-I-V-T-V-V-S-V-L-L-; (k) -S-L-F-V-T-S-V-L-L-C-F-I-; and (I) -M-G-T-Y-G-V-R-A-A-W-R-R-. 4. Recombinant DNA molecule, characterized in that it is able to code ICAM-2 or its functional derivative. 5. A DNA molecule according to claim 4, characterized in that ICAM-2 or its functional derivative is capable of encoding at least one polypeptide from the group comprising: (a) -S-S-F-G-Y-R-T-L-T-V-A-L-; (b) -D-E-K-V-F-E-V-H-V-R-P-K-; (c) -G-S-L-E-V-N-C-S-T-T-C-N-; (d) -H-Y-L-V-S-N-I-S-H-T-D-V-; (e) -S-M-N-S-N-V-S-V-Y-Q-P-P-; (f) -F-T-I-E-C-R-V-P-T-V-E-P-; (g) -G-N-E-T-L-H-Y-E-T-F-G-K-; (h) -T-A-T-F-N-S-T- \ -D-R-E-D-; (i) -H-R-N-F-S-C-L-A-V-L-D-L-; (j) -M-V-I-I-V-T-V-V-S-V-L-L-; (k) -S-L-F-V-T-S-V-L-L-C-F-I-; and (I) -M-G-T-Y-G-V-R-A-A-W-R-R-. 6. Recombinant DNA molecule according to claim 4, characterized in that the molecule is additionally capable of expressing ICAM-2 or its functional derivative in a cell. An antibody or antibody fragment, characterized in that it is capable of binding a molecule selected from the group consisting of ICAM-2 and a functional derivative of ICAM-2. 8. An antibody according to claim 7, characterized in that the molecule is capable of binding to a receptor located on the surface of the cell and due to the binding of the antibody to the molecule, the ability of the molecule to bind to the receptor molecule , impaired, 9. hybridoma, characterized in that it produces the nnonoclonal antibody according to claim. A method of producing a desired hybridoma cell which produces an antibody capable of binding to ICAM-2 or its functional derivative, characterized in that the following steps are carried out: (a) Immunization of an animal with an ICAM-2 expressing cell (b) Fusion of the spleen cells of the animal with a myeloma cell line (c) creating conditions that the fused spleen and myeloma cells produce antibody-secreting hybridoma cells, and (d) screening the hybridoma cells for the desired hybridoma cell capable of producing an antibody capable of binding to ICAM-2. A method of treating inflammation in a mammalian subject characterized in that the subject in need of such treatment is administered an amount of an anti-inflammatory agent sufficient to suppress inflammation, the anti-inflammatory agent being selected from the group consisting of: an antibody, which is able to bind to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; and a non-immunoenibin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 family of molecules. A method according to claim 11, characterized in that the inflammation is a response to a condition selected from the group consisting of: delayed hypersensitivity reaction, psoriatic symptom, autoimmune disease, rejection of an organ or tissue transplants. A method according to claim 12, characterized in that the autoimmune disease belongs to a group comprising the following conditions: Reynaud syndrome, autoimmune thyroiditis, EAE, multiple sclerosis, rheumatoid arthritis and lupus erythematosus. A method according to claim 11, characterized in that the inflammation is in response to a condition belonging to the group comprising: adult respiratory distress syndrome (ARDS), multiple organ damage following septicemia or trauma, myocardial injury or other tissues after elimination of a circulatory disorder, acute glomerulonephritis, secondary arthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other inflammatory disorders of the central nervous system, heat damage, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, syndromes, granulocyte transfusion dia Related and cytokine-induced toxicity. A method for suppressing the metastasis of a tumor cell in the blood-forming tissue, which cell requires a functional member of the CD-18 family for its migration - characterized in that a sufficient amount of an agent to suppress metastasis is administered to a patient in need of such treatment; wherein the agent is selected from the group comprising: an antibody capable of binding to ICAM-2; a toxin-derivatized antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulinantaoonist of ICAM-2 who is not ICAM-Ί or a member of the CD-18 family of molecules. 16. A method for suppressing the growth of an ICAM-2 expressing cell, characterized in that a patient requiring such a treatment is administered an amount of an agent sufficient to suppress the growth, the toxin being selected from the group comprising: an antibody which is able to bind to ICAM-2; a toxin-derivatized antibody capable of binding to ICAM-2; a fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; a toxin-derivatized fragment of an antibody, which fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2; a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 molecule family; a toxin-derivatized member of the CD-18 family of molecules, and a toxin-derivatized functional derivative of a CD-he family of antibodies. A method of suppressing the growth of an LFA-1-expressing tumor cell, comprising administering to a subject in need of such treatment a sufficient amount of toxin to suppress growth, the toxin being selected from the group comprising a toxin-derivatized ICAM. 2 and a toxin-derivatized functional derivative of ICAM-2. A pharmaceutical composition, characterized in that an agent is selected from the group comprising: an antibody capable of binding to ICAM-2; on A fragment of an antibody, wherein the fragment is capable of binding to ICAM-2; ICAM-2; a functional derivative of ICAM-2 and a non-immunoglobulin antagonist of ICAM-2 that is not ICAM-1 or a member of the CD-18 family. 19. Pharmaceutical composition according to claim 18, characterized in that it additionally contains an immunosuppressive agent. 20. Method for detecting the presence of an ICAM-2 expressing cell, characterized in that (a) the cell or an extract of the cell is incubated in the presence of a nucleic acid molecule, wherein the nucleic acid molecule is capable of hybridizing to ICAM-2 mRNA and (b) it is determined whether the nucleic acid molecule has been hybridized to a complementary nucleic acid molecule contained in the cell or in the extract of the cell.