JP2976381B2 - Intercellular adhesion molecule and its binding ligand - Google Patents

Description

DETAILED DESCRIPTION OF RELATED APPLICATIONS This application is filed with US patent application Ser. No. 07 / 045,963 (May 4, 1987).
No. 07 / 115,798 (filed on November 2, 1987), No.
07 / 155,943 (filed February 16, 1988), 07 / 189,815 (filed May 3, 1988), and 07 / 250,446 (filed September 1988).
(Filed March 28) and No. 07 / 324,481 (filed March 16, 1989). This application was made in part with US Government support. The United States Government has certain rights in the invention.

INDUSTRIAL APPLICATION The present invention involves the process in which a group of lymphocytes recognizes and attaches to a cell substrate, penetrates an inflammatory site during an inflammatory reaction and reacts with cells. It relates to an intercellular adhesion molecule such as ICAM-1. The invention further relates to ligand molecules capable of binding to such intercellular adhesion molecules, screening assays for these ligands, and uses of the intercellular adhesion molecules, the ligand molecules and the screening assays.

Prior Art Leukocytes must be able to attach to the cell substrate in order to properly protect the host against enemies such as bacteria or viruses. An excellent view of the defense system is Eisen,
HW " Micro-biology, 3rd edition, published by Harpe & Rows, Philadelphia, PA, (1980), 2
90-295 and 381-418 ". Leukocytes must be able to bind endothelial cells and penetrate the circulatory system to sites of ongoing inflammation. In addition, leukocytes adhere to antigen-presenting cells and become normal. A specific immune response must be able to occur, and further, the lymphocytes must be able to attach to appropriate target cells and cause viral infection or tumor cell degradation.

Recently, leukocyte surface molecules involved in mediating such attachment have been identified using hybridoma technology. In short, human T-cells (Davignon. D. et al., Proc . Natl . Aca
USA 78 : 4535-4538 (1981)] and mouse spleen cells [Springer, T. et al. , Eur. J. Immunol. 9 : 301-306 (197)
9)] Each monoclonal antibody directed thereto was identified as binding to the leukocyte surface and suppressing the above-mentioned adhesion-related functions [Springer. T. et al. , Fed. Proc. 44 : 2660-2 .
663 (1985)]. The molecule identified by these antibodies is M
They are called ac-1 and lymphocyte function associated antigen 1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and granular lymphocytes. LF
A-1 is a heterodimer found in most lymphocytes [Springer. TA et al., Immunol . Rev. 68 : 111-13
5 (1982)]. These two molecules, and the third molecule p15
0,95 (they have similar tissue distribution and Mac-1) exerts a role in cellular adhesion [Keizer, G., Etc., E
ur. J. Immunol. 15 : 1142-1147 (1985)].

The above leukocyte molecules have been found to be members of a related group 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)]. This glycoprotein group is composed of a heterodimer having one alpha chain and one beta chain. Although the alpha chains of each of the antigens are different from each other, the beta chains have been found to be highly protected (Sanchez-Madrid, F. et al., J. Ex .
per. Med. 158 : 1785-1803 (1983)]. The beta chain of glycoproteins (sometimes referred to as "CD18") has been found to have a molecular weight of 95KD, while the alpha chain has a molecular weight of 150K.
D-180 KD (Springer, T. et al.
Et al. , Fed. Proc. 44 : 2660-2663 (1985)]. Although the alpha subunit of the membrane protein does not have the broad homology of the beta subunit, a close analysis of the alpha subunit of the glycoprotein shows that there is substantial similarity between the two. . A study of the similarity between the alpha and beta subunits of LFA-1 related glycoproteins has been made by Sanchez-Madrid, F. et al. [ J.
Exper.Med. 158 : 586-602 (1983); J.Exper.Med.185 :
1785-1803 (1983)].

A group of people who have failed to express a normal amount of any member of the adherent protein group on leukocyte surfaces has been identified [Anderson, DC et al. , Fed. Proc. 44 : 2671-267 .
7 (1985); Anderson, DC et al ., J. Infect . Dis ., 152: 668-
689 (1985)]. The lymphocytes of these patients have the molecular LFA-
One group showed in vivo defects similar to healthy individuals being neutralized by the antibody. Furthermore, the above patients etc.
A normal immune response cannot be measured because their cells cannot adhere to the cell substrate [Anderson, DC et al., F.
ed. Proc. 44 : 2671-2677 (1985); Anderson, DC et al.
J. Infect . Dis ., 152-668-689 (1985)]. These data indicate that the immune response is mitigated when lymphocytes cannot adhere in a normal manner due to the lack of functional adhesion molecules of the LFA-1 family.

Thus, in summary, the ability of lymphocytes to maintain animal health and vitality requires that lymphocytes be able to adhere to other cells (such as endothelial cells). This adhesion has been found to require cell-cell contact involving specific receptor molecules present on the cell surface of lymphocytes. These receptors allow lymphocytes to adhere to other lymphocytes or endothelium and other non-vascular cells. Cell surface receptor molecules have been found to be highly related to each other. Humans whose lymphocytes lack the above cell surface receptor molecules exhibit chronic, recurrent infections, and other clinical symptoms, including defective antibody responses.

Because lymphocyte adherence occurs during the process of recognizing and rejecting foreign tissue, understanding this process is of significant value in the fields of organ transplantation, tissue transplantation, allergy and oncology.

The present invention relates to intercellular adhesion molecule-1 (ICAM-1) and functional derivatives thereof. The present invention further provides antibodies and antibody fragments capable of suppressing the function of ICAM-1, and ICAM
-1 relates to other inhibitors to function, as well as assays that can identify such inhibitors. The invention further relates to diagnostic and therapeutic uses for all of the above molecules.

More specifically, the present invention encompasses the intercellular adhesion molecule ICAM-1 or a functional derivative thereof substantially free of natural impurities. The invention further relates to such molecules capable of binding to molecules present on the surface of lymphocytes.

The invention further relates to a detectably labeled intercellular adhesion molecule IC.
It relates to AM-1 and its derivatives.

The invention further includes a recombinant DNA molecule capable of expressing ICAM-1 or a functional derivative thereof.

The present invention also provides an IC in substantially pure form comprising the following steps:
(A) solubilizing ICAM-1 from a cell membrane expressing ICAM-1 to prepare a solubilized ICAM-1 preparation; and (b) solubilized ICAM. -1 preparation is introduced into an affinity matrix containing an antibody capable of binding to ICAM-1, (c) binding ICAM-1 to an antibody in the affinity matrix, (d) binding to an antibody from the matrix. (E) recovering ICAM-1 in substantially pure form by eluting ICAM-1 from the matrix.

The invention further includes an antibody capable of binding to a molecule selected from the group consisting of ICAM-1 and a functional derivative of ICAM-1. The invention also includes a hybridoma cell capable of producing such an antibody.

The present invention further encompasses a hybridoma cell capable of producing a monoclonal antibody R ₆ -5-D _6.

The present invention further includes a method for producing a desired hybridoma cell producing an antibody capable of binding to ICAM-1, which comprises the following steps.

(A) immunizing an animal with cells expressing ICAM-1; (b) fusing the spleen cells of the animal with a myeloma cell line; (c) antibody-secreting hybridoma cells from the fused spleen and myeloma cells. And (d) screening the hybridoma cells for hybridoma cells capable of producing a desired ICAM-1 binding antibody.

The present invention also includes the hybridoma cells obtained by the above method and antibodies produced from the hybridoma cells.

The present invention also relates to a method of identifying a non-immunoglobulin antagonist of intercellular adhesion, comprising: (a) a plurality of cells capable of aggregating a non-immunoglobulin agent that may be an antagonist of intercellular adhesion. Incubating with a lymphocyte preparation comprising: and (b) examining said lymphocyte preparation to determine whether the presence of said non-immunoglobulin agent inhibits cell aggregation of said lymphocyte preparation; Aggregation inhibition identifies the non-immunoglobulin agent as an antagonist of cell-cell adhesion.

The present invention also relates to a method of treating inflammation resulting from the response of a mammalian specific defense system, the method comprising administering to a subject in need of such treatment an amount of an antibody sufficient to suppress said inflammation. Comprising providing an inflammatory agent, wherein the anti-inflammatory agent comprises an antibody capable of binding to ICAM-1, a fragment of an antibody capable of binding to ICAM-1, ICAM-1, a functional derivative of ICAM-1, and ICAM-1. Are selected from the group consisting of:

The present invention further includes the above-mentioned method for treating inflammation, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.

The present invention also relates to a method for inhibiting metastasis of hematopoietic tumor cells, which requires a functional member of the LFA-1 family for penetration, the method inhibiting such metastasis in a patient in need of such treatment. Providing a sufficient amount of an anti-inflammatory agent to
In addition, the anti-inflammatory agent is an antibody capable of binding to ICAM-1, ICAM-
Fragments of antibodies capable of binding to ICAM-1, ICAM-1,
1 and a non-immunoglobulin antagonist of ICAM-1.

The present invention further includes the above method for inhibiting metastasis of hematopoietic tumor cells, wherein the non-immunoglobulin antagonist of ICAM-1 is a non-immunoglobulin antagonist of ICAM-1 other than LFA-1.

The invention also includes a method of inhibiting the growth of ICAM-1-expressing tumor cells, the method comprising providing a patient in need of such treatment with a sufficient amount of a toxin to inhibit the growth. A toxin-derived antibody capable of binding to ICAM-1, a toxin-derived antibody fragment capable of binding to ICAM-1, a member of the LFA-1 group molecule derived from a toxin, and a member of the LFA-1 group molecule derived from a toxin. It is selected from the group consisting of functional derivatives.

The invention also relates to a method of inhibiting the growth of LFA-1-expressing tumor cells, the method comprising providing a patient in need of such treatment with a sufficient amount of a toxin to inhibit such growth. Including toxin-derived ICAM-1 and IC
It is selected from the group consisting of toxin-derived functional derivatives of AM-1.

The present invention further relates to a method of diagnosing the presence and location of inflammation resulting from the response of a specific defense system in a mammalian subject at risk of having inflammation, the method comprising: (a) providing ICAM-1 to the subject. Administering a composition containing a detectably labeled binding ligand capable of identifying cells that expresses, and (b) detecting the binding ligand.

The invention further relates to a method of diagnosing the presence and location of inflammation resulting from a response of a specific defense system in a mammalian subject at risk of having inflammation, the method comprising: (a) providing a sample of the subject's tissue; Incubating with a composition containing a detectably labeled binding ligand capable of identifying cells expressing ICAM-1; and (b) detecting the binding ligand.

The present invention also relates to a method of diagnosing the presence and location of such cells in a mammalian subject at risk of having ICAM-1-expressing tumor cells, the method comprising: (a) providing the subject with ICAM- Administering a composition comprising a detectably labeled binding ligand capable of binding to 1, wherein said ligand is selected from the group consisting of an antibody and an antibody fragment capable of binding to ICAM-1; and (b) Detecting the binding ligand.

The present invention also relates to a method of diagnosing the presence and location of such cells in a mammalian subject at risk of having ICAM-1-expressing tumor cells, the method comprising: (a) Incubating the sample with a composition containing a detectably labeled binding ligand capable of binding to ICAM-1, wherein the ligand is selected from the group consisting of an antibody and an antibody fragment capable of binding to ICAM-1; and (B) detecting the binding ligand.

The invention also relates to a method of diagnosing the presence and location of such cells in a subject at risk having tumor cells that express a member of the LFA-1 family of molecules, comprising: (a) Administering to the body a composition comprising a detectably labeled binding ligand capable of binding to a member of the LFA-1 family of molecules, wherein the ligand comprises ICAM-1 and ICAM-
(B) detecting the binding ligand.

The invention also relates to a method of diagnosing the presence and location of such cells in a subject at risk having tumor cells that express a member of the LFA-1 family of molecules, comprising: (a) A sample of body tissue was used for molecular LFA-1
Incubating in the presence of a detectably labeled binding ligand capable of binding a member of the group, wherein the ligand comprises
Being selected from the group consisting of ICAM-1 and functional derivatives of ICAM-1, and (b) LFA-1 of the molecule present in the tissue sample
Detecting said binding ligand bound to a member of the group.

The present invention further provides: (a) antibodies capable of binding to ICAM-1, fragments of antibodies capable of binding to ICAM-1, ICAM-1, functional fragments of ICAM-1, and non-immunoglobulin antagonists of ICAM-1 And (b) at least one immunosuppressive agent selected from dexamezone, azathiopyrine and cyclosporin A.

Also included are pharmaceutical compositions comprising

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically shows the adhesion between normal cells and LFA-1 deficient cells.

FIG. 2 schematically shows the normal cell / normal cell adhesion process.

FIG. 3 shows the kinetics of cell aggregation in the absence (×) or presence (○) of 50 ng / m PMA.

Figure 4 is LFA-1 ^- shows the aggregation between cells and LFA-1 ⁺ cells.
As shown in the figure, carboxyfluorescein diacetate-labeled EBV-transformed cells (10 ⁴ ) were mixed with 10 ⁵ unlabeled allogeneic cells (black frame) or JY cells (white frame) in the presence of PMA. After 1.5 hours, the labeled cells in aggregates or free were counted using the quantitative assay of Example 2. Shows the percentage of labeled cells in the aggregate. One representative test of the two is shown.

FIG. 5 shows immunoprecipitation of ICAM-1 and LFA-1 from JY cells. Antibodies that can bind Triton X-100 lysate of JY cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) to ICAM-1 (lanes 1 and 3)
Or an antibody capable of binding to LFA-1 (lanes 2 and 4)
For immunoprecipitation. Panel A shows the results under reducing conditions and panel B shows the results obtained under non-reducing conditions. Molecular weight standards are shown in lane S.

FIG. 6 shows the kinetics of the effects of LFA-1 and gamma interferon on ICAM-1 expression on human dermal fibroblasts. Human skin fibroblasts are 8 × 10 ⁴ cells / 0.32 cm ²
Propagated to the density of the well. IL-1 (10μ / m, black circle)
Alternatively, recombinant gamma interferon (10 μ / m, open square) was added, and the mixture was cooled to 4 ° C. for the time shown in the figure, and an indirect binding assay was performed. Standard deviation did not exceed 10%.

FIG. 7 shows the concentration dependence of the effects of IL-1 and gamma-interferon on ICAM-1. 8 human skin fibroblasts
The cells were grown to a density of × 10 ⁴ cells / 0.32 cm ₂ well. IL-2
(Open circle), recombinant human IL-1 (open square), recombinant mouse IL
-1 (closed square) and recombinant beta interferon (open triangle) at the dilutions shown in the figure for 4 hours (IL-1) or 16 hours (beta and gamma interferon)
Incubated. The results shown represent the average of four measurements, with a standard deviation not exceeding 10%.

FIG. 8 shows the nucleotide and amino acid sequence of ICAM-1 cDNA. The first ATG is at position 58. The translated sequence corresponding to the ICAM-1 trypsin peptide is underlined. The putative hydrophobicity signal peptide and the transmembrane sequence are underlined in bold. N-
The binding glycosylation site is boxed. The polyadenylation signal AATAAA at position 2976 is overlined. The sequence shown is for the HL-60 cDNA clone. Endothelial cell cDNA was sequenced over most of its length and showed only small differences.

FIG. 9 shows the correlation to the ICAM-1 homology domain and immunoglobulin supergenes. (A) Sequence of five homologous domains (D _1-5 ): Two or more identical residues in a row are boxed. Residues contained in the residues included more than once in NCAM domains, and a set C ₂ and C ₁ domains are arranged by ICAM-1 internal repeats. The position of the predicted β-stride in the domain of ICAM-1 is marked with a bar and a lowercase letter on the sequence, and the known position of the β-strand in the immunoglobulin c domain is marked with a bar and a capital letter below the sequence. doing. The location of the putative disulfide bridge within the ICAM-1 domain is indicated by SS. ICAM−
(BD) sequence of protein domains homologous to 1: Each protein is first sequenced by searching the NBRF database using the FASTP program. Each protein sequence is
MAG, NCAN, T cell receptor α subunit V domain, IgM μ chain, and α-1-B glycoprotein.

 FIG. 10 is a comparison diagram of the secondary structure between ICAM-1 and MAG.

FIG. 11 shows LFA-1 bound to ICAM-1 in the flat film.
Positive EBV-transformed B-lymphoblastoma
d) Indicates cells.

FIG. 12 shows LFA-1 positive T-lymphoblasts and T-lymphocytes bound to ICAM-1 in plastic-bound vesicles.

FIG. 13 shows the inhibition of binding of JYB-lymphoblastoma cells or vesicles bound to ICAM-1 in plastic-bound vesicles by pretreatment with monoclonal antibodies.

FIG. 14 shows the effect of temperature on T-lymphoblast binding to ICAM-1 in plastic-bound vesicles.

FIG. 15 shows the need for divalent cations in binding T-lymphoblasts to ICAM-1 in plastic-bound vesicles.

FIG. 16 shows the effect of anti-adherent antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the T-cell associated antigen OKT3. "OKT3" indicates addition of antigen.

FIG. 17 shows the effect of anti-adherent antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the nonspecific T-mitogen, Concanavalin A. “CO
NA "indicates the addition of concanavalin A.

FIG. 18 shows the effect of anti-adherent antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of keyhole limpet hemocyanin antigen. "KLH" indicates the addition of keyhole limpet hemocyanin to cells.

FIG. 19 shows the effect of anti-adhesive antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of tetanus toxoid antigen. “AGN” indicates the addition of tetanus toxoid antigen to cells.

FIG. 20 shows a monoclonal antibody against the ICAM-1 deletion mutant
FIG. 9 shows binding of RR1 / 1, R6.5, LB2, and CL203.

FIG. 21 shows the binding of the ICAM-1 deletion mutant to LFA-1.

FIG. 22 shows anti-ICAM-1 monoclonal antibodies RR1 / 1, R6.
5, epitopes recognized by LB2 and CL203.

FIG. 23 shows the binding ability of the ICAM-1 domain 2 mutant to LFA-1.

FIG. 24 shows the binding ability of the ICAM-1 domain 3 mutant to LFA-1.

FIG. 25 shows the binding ability of the ICAM-1 domain 1 mutant to LFA-1.

 FIG. 26 shows the sequence of the ICAM amino-terminal domain.

Preferred Embodiments One aspect of the present invention relates to the discovery of a natural binding ligand for LFA-1. Molecules such as LFA-1 family molecules are involved in the process of cell-cell adhesion and are referred to as "adhesion molecules".

The natural binding ligand of the present invention is "cell adhesion molecule-1".
(Intercellular Adhesion Molecule-1) ”or“ I
CAM-1 ". ICAM-1 is a 76-97 kd glycoprotein. ICAM-1 is not a heterodimer. The present invention relates to ICAM-1 and its" functional derivatives ".
A "functional derivative" of -1 is a compound having a biological activity (functional or structural) substantially similar to that of ICAM-1. The term "functional derivative" is intended to include "fragments,""variants,""homologs" or "chemical derivatives" of a molecule. A "fragment" of a molecule, such as ICAM-1, refers to any polypeptide subset of the molecule. Fragments of ICAM-1 that have ICAM-1 activity and are soluble (ie, not membrane bound) are particularly preferred. A “variant” of a molecule, such as ICAM-1, refers to a molecule that is substantially similar in structure or function to the entire molecule or a fragment thereof. A molecule is said to be "substantially similar" if both molecules have a substantially similar structure to the other molecule or if both molecules have similar biological activity. That is, when two molecules have similar activities, the molecules are considered to be variants, and the term is used herein when one structure of the molecule is not found in the other molecule or when the amino acid residue is present. It is used even when the base sequences are not the same. An "analog" or "homolog" of a molecule such as ICAM-1 refers to a molecule that is substantially similar in function to the entire molecule or a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule if the molecule normally contains additional chemical moieties that are not part of the molecule. Such components may improve the solubility, absorption, biological half-life, etc. of the molecule. These components also reduce the toxicity of the molecule and eliminate or attenuate unwanted side effects of the molecule. An ingredient that can mitigate such effects is "Remington's Pharmaceutical
utical Sciences (1980). “Toxin-derived” molecules constitute a special class of “chemical derivatives.” “Toxin-derived” molecules are those molecules that have a toxin component (ICAM).
-1 or antibody). The binding of such molecules to cells carries the toxin component in close proximity to the cells, thereby promoting cell death. Although any suitable toxin component can be used, it is preferred to use toxins such as, for example, ricin toxin, diphtheria toxin, radioisotope toxin, and membrane-channel forming toxin. Methods for attaching such components to molecules are well known in the art.

Antigenic molecules, such as ICAM-1 or a member of the LFA-1 family of molecules, are naturally expressed on lymphocyte surfaces. That is, introduction of such cells into an appropriate animal, such as by intraperitoneal injection, will result in the production of antibodies capable of binding to a member of the ICAM-1 or LFA-1 family of molecules. In some cases, the serum of such animals can be removed and used as a source of polyclonal antibodies capable of binding to these molecules. However, preference is given to removing spleen cytes from such animals, fusing such spleen cells with a myeloma cell line, and obtaining ICAM from the resulting fused cells.
To establish a hybridoma cell producing a monoclonal antibody capable of binding to a member of the LFA-1 or LFA-1 family of molecules.

The hybridoma cells obtained by the above method can be screened by various methods to identify the desired hybridoma cells that produce antibodies capable of binding to either ICAM-1 or a member of the LFA-1 group of molecules. In one preferred screening assay, such molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus transformed cells. Antibodies capable of inhibiting such aggregation are further screened to determine whether these antibodies inhibited such aggregation by binding to a member of the ICAM-1 or LFA-1 family of molecules. ICAM−
Any means that can distinguish 1 from LFA-1 group molecules can be used for such screening. For example, antigen bound to antibodies can be analyzed as by immunoprecipitation and polyacrylamide gel electrophoresis. Bound antigen
If it is a member of the LFA-1 family of molecules, the immunoprecipitated antigen will be found as a dimer, while
If the binding antigen is ICAM-1, a single molecular weight species will be immunoprecipitated. Further, an antibody that binds to a member of the LFA-1 group molecule is different from an antibody that binds to ICAM-1 from LFA-1.
Can also be distinguished by screening for the ability of the antibody to bind to cells such as granulocytes that do not express ICAM-1. The ability of an antibody (known to inhibit cell aggregation) to bind to granulocytes depends on the ability of the antibody to
-1. Absence of such binding may indicate an antibody that can recognize ICAM-1. The ability of an antibody to bind to cells, such as granulocytes, can also be detected by methods commonly used by those of ordinary skill. Such methods include immunoassays, cell aggregation, filter binding tests, antibody precipitation, and the like.

The anti-aggregated antibodies of the present invention also determine their ability to specifically bind to cells expressing ICAM-1 (such as activated endothelial cells) and their inability to bind to cells that do not express ICAM-1. Can also be identified. As will be readily appreciated by those skilled in the art, each of the above assays can be modified, i.e., performed in a different order, to provide a variety of potential screening assays, each of which has an IC
It can identify and differentiate between an antibody capable of binding to AM-1 and a member of the LFA-1 group of molecules.

The anti-inflammatory agent of the present invention may be a polyclonal antibody capable of binding animals to ICAM-1 by inducing natural methods (eg, animals, plants, fungi, doctoria, etc.) to produce non-immunoglobulin antagonists of ICAM-1. ), Such as by inducing to produce ICAM-1, a functional derivative of ICAM-1 or ICAM-1 using the Merrifield method for polypeptide synthesis.
With protein antagonists of AM-1 (such as by synthesizing either immunoglobulins or non-immunoglobulins), hybridoma technology (eg, ICAM-
1) or by recombinant techniques (eg, the anti-inflammatory agents of the present invention can be used in a variety of hosts (eg, yeast, bacteria, fungi, cultured animal cells, etc.), or The choice of the method used depends on factors such as convenience, desired yield, etc. Identification using only one of the methods, techniques or techniques described above. No need to produce anti-inflammatory drugs,
The above methods, techniques and techniques may be combined to obtain specific anti-inflammatory agents.

A. Identification of LFA-1 binding partner (ICAM-1) 1. Assay for LFA-1 dependent aggregation Many Epstein-Barr virus transformed cells show aggregation. This aggregation can be promoted in the presence of the phorbol ester. Such homotypic aggregation (ie, aggregation involving only one cell type) has been found to be blocked by anti-LFA-1 antibodies [“Rothlei
J. Exper. Med . 163 : 1132-1149 (1986), "which is incorporated herein by reference.] That is, LFA-1
The degree of dependent binding can be determined by assessing the degree of spontaneous or phorbol ester-dependent aggregation formation.

Agents that interfere with LFA-1-dependent aggregation can be identified by using an assay that can determine whether the agent inhibits spontaneous or phorbol ester-dependent aggregation of Epstein-Barr virus transformed cells. Most Epstein-Barr virus transformed cells can be used in such assays as long as they can express the LFA-1 receptor molecule. Such cells are described in "Springer, TA et al., J. Exper . Med . 160: 1901-1918 (198
4) ", which is incorporated herein by reference. Any such cells may be prepared according to the present invention.
Although it can be used in an LFA-1-dependent binding assay, it is preferred to use cells of the JY cell line [Terhost, CT et al ., Proc. Natl. Acad. Sci. USA, 73 : 910 (1
976)]. These cells can be cultured in any suitable culture medium, most preferably 10% fetal bovine serum and 50%
μg / m gentamicin (Gib Laboratories, Inc.
Culturing the cells in RMP11640 medium (New York). These cells should be cultured under conditions suitable for animal cell growth (ie, generally at a temperature of 37 ° C., in an atmosphere of 5% CO ₂ , at a relative humidity of 95%, etc.).

2. Binding of LFA-1 to ICAM-1 Human individuals whose lymphocytes lack a group of LFA-1 receptor molecules have been identified [Anderson, DC et al. , Fed. Proc.
44 : 2671-2677 (1985); Anderson, DC et al., J. Infect . Di.
s.152 : 668-689 (1985)]. Such people are said to suffer from leukocyte adhesion deficiency (LAD). Such human EBV transformed cells do not aggregate either spontaneously or in the presence of phorbol esters in the agglutination assays described above. Aggregation is observed when such cells are mixed with LFA-1 expressing cells [Rothlein, R. et al., JE .
xper.Med.163 : 1132-1149 (1986)] (FIG. 1). Importantly, these aggregates did not form when these cells were incubated in the presence of anti-LFA-1 antibody. That is, aggregation required LFA-1 but L
The ability of FA-1 deficient cells to form aggregates with LFA-1-containing cells suggested that the LFA-1 binding partner was not LFA-1, but rather a previously undiscovered intercellular adhesion molecule.
FIG. 1 shows the mechanism of cell-cell adhesion.

B. Intercellular adhesion molecule-1 (ICAM-1) A novel intercellular adhesion molecule ICAM-1 is described in "Rothlein, R., et al . [ J. Immunol . 137 : 1270-1274 (1986); To detect the ICAM-1 molecule, a monoclonal antibody was immunized with human cells genetically deficient in LFA-1 expression to detect the ICAM-1 molecule. The resulting antibodies were screened for their ability to inhibit the aggregation of LFA-1 expressing cells (FIG. 2).
Specifically, ICAM-1 molecule mice were immunized with EBV-transformed B cells from a LAD patient that did not express the LFA-1 antigen. Spleen cells from these animals were removed and fused with myeloma cells to produce monoclonal antibody-producing hybridoma cells. Next, EBV-transformed B cells from a normal human expressing LFA-1 were incubated in the presence of the hybridoma cell monoclonal antibody described above to induce phorbol ester-mediated, LFA-1-dependent, spontaneous expression of EBV transformed B cells. Any monoclonal antibody capable of inhibiting aggregation was identified. Since the above hybridoma cells were derived from cells containing no LFA-1 antigen, no monoclonal antibodies against LFA-1 were produced. Thus, any antibody found to inhibit aggregation must be capable of binding to an antigen that is not LFA-1, but was involved in the LFA-1 adhesion process. Although any method can be used to obtain such a monoclonal antibody, BALB / C mice are preferably obtained from Rothlein, R.
ICAM by immunizing with Epstein-Barr virus-transformed peripheral blood mononuclear cells from LFA-1 deficient using the route and schedule disclosed by J. Immunol. 137: 1270-1274 (1986). -1 binding monoclonal antibody. Such cells are Sp
ringer, TA, etc. [ J.Exper.Med.16
0 : 1901-1918 (1984)].

In a preferred method for the production and detection of antibodies capable of binding to ICAM-1, mice are treated with ICAM-1 and LFA
-1 is immunized with EBV-transformed B cells expressing both, or more preferably with TNF-activated endothelial cells that express ICAM-1 but do not express LFA-1. Anti-ICAM-
In the most preferred method of preparing a hybridoma cell producing one antibody, Balb / C mice are sequentially immunized with JY cells and specific U937 cells (ATCC CRL-1593). Spleen cells from such animals are removed and fused with myelor cells to develop antibodies into productive hybridoma cells. The obtained antibody was used for its LFA-1 and ICAM-1.
L of EBV-transformed cell lines such as JY cells expressing both
Screen for ability to inhibit FA-1 dependent, phorbol ester-induced aggregation. Rothlein, R., etc. ( J.Imm
unol. 137 : 1270-1274 (1987)], an antibody capable of controlling such aggregation was identified as SKW ₃ [Dust
In, M. et al., J. Exper. Med . 165 : 672-692 (1987), whose ability to aggregate spontaneously in the presence of phorbol esters is LF.
Suppressed by antibodies capable of binding to A-1, but anti-ICAM
-1 antibody is not inhibited] is tested for its ability to inhibit phorbol ester-induced aggregation. Antibodies formate capable of inhibiting the ball ester induced aggregation of cells such as JY cells, but incapable of inhibiting the phorbol ester induced aggregation of cells such as SKW ₃ is probably anti-ICAM-1 antibody. In addition, an antibody capable of binding to ICAM-1 can suppress LFA-1-dependent aggregation of LFA-expressing cells (such as JY cells), but almost completely expresses LCAM-1. Or an antibody that cannot bind to cells that do not express at all (such as normal granulocytes) or an antibody that expresses ICAM-1 can bind to cells that do not express LFA-1 (such as TNF-activated endothelial cells) Can also be identified by screening. Another method is to immunoprecipitate cells that express ICAM-1, LFA-1, or both using an antibody that inhibits LFA-1-dependent aggregation of cells such as JY cells and perform SDS-PAGE or equivalent. The method is to determine some molecular properties of the molecules precipitated by the antibody. If the properties are the same as those of ICAM-1, the antibody can be considered to be an anti-ICAM-1-antibody.

Using the monoclonal antibody prepared by the method described above,
The ICAM-1 cell surface molecule was purified and characterized. ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. In such a method, a monoclonal antibody reactive with ICAM-1 is bound to an inert column matrix. Although any method of performing such conjugation can be used, preferred is "Oetthen, HC et al., J. Biol . Chem. 259: 12034 (1984)".
Is used. When the cell lysate is passed through the matrix, the ICAM-1 molecules present are adsorbed and retained on the matrix. By changing the pH or ionic concentration of the column, bound ICAM-1 molecules can be eluted from the column. Although any suitable matrix can be used, it is preferred to use Sepharose (Pharmacia) as the matrix material. The preparation of column matrices and their use in protein purification is well known in the art.

In a manner understood by those skilled in the art, the assays described above can be used to identify compounds that can attenuate or suppress the rate or extent of cell adhesion.

ICAM-1 is found on vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and on non-hematopoietic cells such as fibroblasts and on tissue macrophages, mitogen-stimulated T lymphoblasts, germinal center B in tonsils. Cell surface glycoproteins expressed on cells and hematopoietic cells such as dendritic cells, lymph nodes, and Peyer's patches. ICAM-1 is highly expressed on vascular endothelial cells in T cell areas within lymph nodes and tonsils showing reactive proliferation. ICAM-1 is expressed in small amounts on peripheral blood lymphocytes. Phorbol ester-stimulated specification of certain bone monocyte cell lines greatly increases ICAM-1 expression. That is, ICAM-1 is preferentially expressed at sites of inflammation and is not generally expressed in quiescent cells, but on skin fibroblasts.
ICAM-1 expression is at a level of 10 μ / m interleukin-1
Alternatively, gamma-interferon increases 3 to 5 times in 4 hours or 10 hours or more, respectively. Its induction depends on protein and mRNA synthesis and is reversible.

ICAM-1 exhibits molecular weight heterogeneity in different cell tumors with a molecular weight of 97 Kd on fibroblasts, 114 Kd on osteoblastic monocytic cell lines, and 90 Kd on B lymphoblastoid cells JY. ICAM-1 biosynthesis has been shown to involve an intercellular precursor of about 73 Kd. The non-N-glycosylated form obtained from tunicamycin treatment (suppresses glycosylation) has a molecular weight of 55 Kd.

ICAM-1 isolated from phorbol ester-stimulated U937 cells or from fibroblast cells is 60-fold after chemical deglycosylation.
This gives the same major product with a molecular weight of Kd. ICAM−
1 Monoclonal antibody is LF of phytohemagglutinin blasts
Interferes with adhesion to A-1 deficient cell lines. Pretreatment of fibroblasts (but not lymphocytes) with monoclonal antibodies conjugated to ICAM-1 suppresses lymphocyte-fibroblast adhesion. Pretreatment of lymphocytes with antibodies to LFA-1 (without fibroblasts) has also been found to suppress lymphocyte-fibroblast adhesion.

Thus, ICAM-1 is a binding ligand for the CD18 complex on leukocytes. ICAM-1 is time-coordinated with lymphocyte infiltration into inflammatory areas in vivo on fibroblasts and endothelial cells by inflammatory mediators such as IL-1, gamma-interferon and tumor necrosis factor. Inducible (Dustin, ML et al. , J. Immunol . 137 : 245
−254, (1986): Prober, JS, et al. , J. Immunol. 137: 1893
-1896, (1986)]. In addition, ICAM-1 is found on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells and fibroblasts and also on tissue macrophages, mitogen-stimulated T lymphoblasts, germinal center B cells in tonsils. And also on hematopoietic cells such as dendritic cells, lymph nodes and Peyer's patches (Dustin, ML et al. , J. Immunol . 137 : 2
45-254, (1986)]. ICAM-1- is also expressed on benign inflamed keratinocytes such as allergic eczema, lichen planus, rash, urticaria and vesicular disease. Allergic skin reactions induced by the application of haptens to the skin of allergic patients also resulted in high levels of ICAM-1 expression on keratinocytes. In contrast, the toxic patch on the skin did not result in ICAM-1 expression on keratinocytes. ICAM-1 is present on keratinocytes from biopsies of skin lesions from various dermatological irregularities, and ICAM-1 expression was induced in disorders from allergy patch tests, but keratinocytes from toxic patch test disorders. Is ICAM−
1 was not expressed.

ICAM-1 is therefore a cellular substrate to which lymphocytes can attach, so that lymphocytes can penetrate sites of inflammation and / or transmit various effector functions that contribute to this inflammation. Such functions include production of antibodies, lysis of virus infected target cells, and the like. As used herein, the term "inflammation" is meant to include a specific or non-specific defense system response. The term "specific defense system" shall refer to a component of the immune system that reacts with the presence of a characteristic antigen. Inflammation is said to be caused by, mediated by, or accompanied by a response of the specific defense system, resulting from a response of the specific defense system. Examples of inflammation resulting from a specific defense system response include rubella virus, autoimmune diseases, delayed-type hypersensitivity mediated by T cells (eg, in patients who test "positive" in the Mantaux test), psoriasis, etc. There is.

A "non-specific defense system response" is a response mediated by leukocytes that is unable to memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to result from a non-specific defense system response if the inflammation is caused, mediated, or accompanied by a response of the non-specific defense system. .
Examples of inflammation at least in part derived from the non-specific defense system include asthma, adult respiratory distress syndrome (ARDS) or septic or traumatic secondary organ damage groups, reperfusion of myocardium or other tissues (Reperfusion) injury, acute glomerulonephritis,
Reactive arthritis, dermatitis due to acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory diseases, burns, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing colitis There are inflammations associated with conditions such as, granulocyte transfusion-related syndrome, and cytokine-induced toxicity.

According to the present invention, ICAM-1 functional derivatives, particularly those comprising fragments or mutants of ICAM-1 having domains 1, 2 and 3, are used to treat non-specific defense system reactions as described above. Or it can be used in therapy.
More preferred in such treatment or therapy is IC
An ICAM-1 fragment or mutant containing domain 2 of AM-1. Most preferred for such treatment or therapy contains domain 1 of ICAM-1
ICAM-1 fragment or mutant. Furthermore, the present invention contemplates the use of ICAM-1 and functional derivatives thereof in the treatment of asthma.

C. Cloning of ICAM-1 Gene The ICAM-1 gene can be cloned using any of a number of methods. One such method is to analyze a shuttle vector library of cDNA inserts (from ICAM-expressing cells) for the presence of an insert containing the ICAM-1 gene. Such an analysis can be performed by transfecting cells with the above vector and assaying for ICAM-1 expression. A preferred method of cloning this gene involves determining the amino acid sequence of the ICAM-1 molecule. To do this, the ICAM-1 protein can be purified and analyzed by an automated sequenator. Also,
The molecule can be degraded by cyanogen bromide or by a protease such as papain, chymotrypsin or trypsin [Oike, Y. et al., J. Biol . Chem . 257: 9751.
-9758 (1982): Liu, C. et al., Int, J. Pept . Protein Res.2
1 : 209-215 (1983)]. Although the entire amino acid sequence of ICAM-1 can be determined, it is preferable to determine the sequence of the peptide fragment of the molecule. If the peptide is longer than 10 amino acids, the sequence information is generally ICAM-1
Is sufficient to clone a gene such as the gene of

The sequence of amino acid residues in the peptide is also indicated herein using commonly used three-letter or one-letter codes. A list of these three-letter and one-letter representations can be found in " Biochemistry , Lehninger, A., Worth Publishers,
New York, (1970) ". When writing such sequences vertically, the amino end groups should be at the top of the row and the carboxy end groups should be at the bottom of the row. When written horizontally, the amino terminus is on the left and the carboxy terminus is on the right. Amino acid residues in the peptide can be separated by a hyphen. It is only intended to be easy, just to illustrate: The amino acid sequence indicated as A indicates that the Aa residue is bonded to the carboxy group of Gly and that the Ser residue is bonded to the carboxy group of the Aa residue and the amino group of the Phe residue. The representation also shows that the amino acid sequence contains the tetrapeptide Gy-Aa-Ser-Phe. This notation does not limit the amino acid sequence to this one tetrapeptide; (1) a tetrapeptide having one or more amino acid residues attached to an amino or carboxy, (2) both amino and carboxy termini. It is intended to include tetrapeptides having one or more amino acid residues linked thereto, and (3) tetrapeptides having no additional amino acid residues.

Once one or more appropriate peptide fragments have been sequenced, the DNA sequences that can encode them are examined. Since the genetic code is degenerate, one or more codons can be used to encode a particular amino acid [Watsno, JD, Mo
lecular Biology of the Gene, 3rd edition, WABenjamin
Company, California Menlo Park, (1977), pp356
−357]. The peptide fragments are analyzed to identify the amino acid sequence that can be encoded by the oligonucleotide with the least degree of degeneracy. This is preferably done by identifying sequences containing amino acids encoded by only a single codon. In some cases, such an amino acid sequence may be encoded by only a single oligonucleotide, but in many cases, the amino acid sequence may be encoded by any one set of similar oligonucleotides. Importantly, all members of the set contain oligonucleotides capable of encoding the peptide fragment, and thus potentially contain the same nucleotide sequence as the gene encoding the peptide fragment, whereas one member of the set described above Only contains the same nucleotide sequence as the nucleotide sequence of this gene. Since this member is present in the set and can hybridize to DNA even in the presence of other members of the set, it is in the same manner as using a single oligonucleotide to clone the gene encoding the peptide. Undegraded oligonucleotide sets can be used.

In exactly the same manner as described above, an oligonucleotide (or set of oligonucleotides) having a nucleotide sequence that is complementary to an oligonucleotide sequence or set of sequences capable of encoding a peptide fragment may be used.

A suitable oligonucleotide or set of oligonucleotides (or those that are complementary to such oligonucleotides or oligonucleotides) capable of encoding a fragment of the ICAM-1 gene is identified (using the procedures described above) and the DNA It is preferably synthesized and hybridized with a human cell-derived cDNA preparation capable of expressing the ICAM-1 gene sequence using means well known in the art.
Nucleic acid hybridization technology is described in “Mol
ecular Cloning, a Laboratory Manual, Coldspring Harb
or, NY (1982) ", and by Haymes, BD, etc.," Nucleic acid Hybrization, a Practical approach, IR
L Press, Washington, DC (1985) ", which are hereby incorporated by reference. The DNA or cDNA source used is preferably rich in the ICAM-1 sequence. It can be most easily obtained from cDNA obtained by extracting RNA from cells cultured under conditions that induce ICAM-1 synthesis (such as U937 grown in the presence of phorbol ester).

Genes such or similar methods human aldehyde dehydrogenase as the methods described above [Hsu, LC, etc., Pro
c, Natl, Acad, Sci, USA 82 : 3771-3775 (1985)], fibronectin [Suzuki, S. et al ., Eur, Mol, Biol, Organ, J.4 :
2519-2524 (1985)] human estrogen receptor
Gene (Walter, P. et al., Proc, Natl, Acad, Sci, USA 82 : 7889
-7993 (1985)], tissue-type plasminogen activator [Pennica, D. et al., Nature 301 : 214-221 (1983)] and human placental alkaline phosphatase complementary DNA [Kam,
W. et al., Proc, Natl, Acad, Sci, USA 82 : 8715-8719 (198
5)] has been successfully achieved.

In another preferred method of cloning the ICAM-1 gene, a library of expression vectors is prepared by cloning DNA or, preferably, cDNA from cells capable of expressing ICAM-1 in the expression vector. This library was then bound to anti-ICAM-1 antibody and
One is screened for a member capable of expressing a protein having a nucleotide sequence capable of encoding a polypeptide having the same amino acid sequence as the ICAM-1 fragment.

The cloned ICAM-1 gene obtained by the above method is ligated to an expression vector by an operation and incorporated into bacterial or eukaryotic cells to produce ICAM-1 protein. Such manipulation techniques are disclosed by Maniatis, T. et al., Supra, and are well known in the art.

D. Using Assays for LFA-1-Dependent Aggregation The assays described above that can measure LFA-1-dependent aggregation can be used to identify agents that act as antagonists that inhibit the extent of LFA-1-dependent aggregation. Such antagonists may act by conferring the ability of LFA-1 or ICAM-1 to participate in aggregation. That is, such agents include immunoglobulins such as antibodies capable of binding LFA-1 or ICAM-1. In addition, non-immunoglobulin (ie, chemical) agents can also be used to determine whether they are antagonists of LFA-1 aggregation using the assays described above.

E. ICAM-1 using an antibody capable of binding to a receptor protein 1. Binding of monoclonal antibodies against each one member of the anti-inflammatory agent CD ₁₈ complexes into endothelial [Haskard, D, etc., J.Immunol.137:. 2901 −2906
(1986)], homotypic adhesion [Rothlein, R. et al., J. Exp . M.
ed. 163 : 1132-1149 (1986)] Antigen and mitodin-induced proliferation of lymphocytes [Davigon, D. et al., Proc, Natl, Acad, S.
ci, USA 78 : 4535-4539 (1981)], antibody formation [Fisher, A.
J. Immunol. 136: 3198-3203 (1986)], and cytotoxic T cells [Krensky, AM et al. , J. Immunol. 132: 2180-21].
82 (1984)], macrophages [Strassman, G., et al., J. Im .
munol. 136 : 4328-4333 (1986)], and all cells involved in the antibody-dependent cytotoxicity reaction [Kohi, S. et al., J. Immun .
ol. 133 : 2972-2978 (1984)], which inhibits many adhesion-dependent functions of leukocytes, including all leukocyte effector functions. In all of these functions, the antibodies inhibit the ability of leukocytes to adhere to the appropriate cell substrate, which ability suppresses the end result.

As described above, one of the LCAM-1 group of ICAM-1 molecules
Binding to members is crucial in cell adhesion. During the process of adherence, lymphocytes can continuously monitor animals for the presence of foreign antigens. Although these processes are usually desirable, they are also responsible for organ transplant rejection, tissue transplant rejection, and many autoimmune patients. Therefore, some means of attenuating or suppressing cell adhesion is highly desirable for recipients of autologous or organ transplants or tissue transplants.

Monoclonal antibodies capable of binding to ICAM-1 are extremely suitable as mammalian anti-inflammatory agents. Clearly, such drugs differ from common anti-inflammatory drugs in that they can selectively inhibit sticking and do not exhibit other side effects such as nephrotoxicity that can be found with conventional drugs. Thus, monoclonal antibodies capable of binding to ICAM-1 can be used to prevent organ or tissue rejection or modify the autoimmune response in mammals without fear of such side effects.

Importantly, the use of monoclonal antibodies capable of recognizing ICAM-1 allows organ transplantation to be performed even in individuals with HIA imbalance.

2. Suppressor of delayed-type hypersensitivity reaction Since ICAM-1 molecule is almost expressed in inflammatory sites such as those involved in delayed-type hypersensitivity reaction, antibodies (particularly monoclonal antibodies) that can bind to ICAM-1 molecule are Have therapeutic potential in attenuating or eliminating such responses. This potential therapeutic application can be exploited in one of two ways. First, a composition containing a monoclonal antibody to ICAM-1 can be administered to a patient exhibiting a delayed type hypersensitivity reaction. For example, such a composition could be administered to a person who has been contacted with an antigen, such as a poisonous fox, a poisonous oak, or the like. In a second embodiment, a monoclonal antibody capable of binding to ICAM-1 is administered to the patient along with the antigen to prevent a subsequent inflammatory response. That is, co-administration of an antigen and an ICAM-1-binding monoclonal antibody can be temporarily tolerated by humans after administration of the antigen.

3. Treatment of Chronic Inflammatory Disease Since LAD patients deficient in LFA-1 do not show an inflammatory response, it is believed that antagonism of the LFA-1 natural ligand, ICAM-1, also suppresses the inflammatory response. The ability of antibodies against ICAM-1 to suppress inflammation is indicated by discoid erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some aspects of diabetic Reynard's syndrome, rheumatoid It provides a basis for therapeutic use in the treatment of chronic inflammation and autoimmune diseases such as arthritis. In general,
Monoclonal antibodies capable of binding ICAM-1 can be used to treat diseases currently treatable by steroid therapy.

4. Diagnostic and Deterministic Uses Since ICAM-1 is expressed mostly in inflammatory areas, ICAM-1
Can be used as a means to image or visualize infected and inflammatory sites in a patient. In such applications, the monoclonal antibodies may be radioisotopes, affinity labels (biotin,
Avidin) (eg, avidin), fluorescent labels, paramagnetic atoms and the like. Procedures for performing such labeling are well known in the art. The clinical application of antibodies in diagnostic imaging is “Grossman, HB, Urol, Clin, Nort.
h Amer. 13 : 465-474 (1986) "," Unger, EC, etc., Inve.
st, Radiol, 20 : 693-700 (1985) "and" Khaw, BA
Et al., Science 209 : 295-297 (1980) ".

The presence of inflammation is also dependent on ICAM-1 expression in cells expressing ICAM-1.
MRNA, c that binds to the gene sequence or to the ICAM-1 mRNA sequence
Detection can also be by the use of DNA or a binding ligand such as DNA. Techniques for performing such a hybridization assay are disclosed by Manitaitas, T. (supra).

Detection of a lesion of such a detectably labeled antibody may indicate a site of inflammation or tumor development. In one embodiment, the inflammation test is performed by removing a sample of tissue or blood and incubating such a sample in the presence of the detectably labeled antibody. In a preferred embodiment, the method comprises magnetic imaging,
This is performed in a non-invasive manner using fluorography or the like. Such diagnostic tests can be used to monitor organ transplant recipients for early detection of potential tissue rejection. Such assays can also be performed to determine an individual's response to rheumatoid arthritis or other chronic inflammatory diseases.

5. Assist in the introduction of antigenic substances to be administered for therapeutic or diagnostic purposes. For example, an immune response to a therapeutic or diagnostic agent such as bovine insulin, interferon, tissue-type plasminogen activator or mouse monoclonal antibody may It can substantially reduce the value of treatment or diagnosis and, in fact, cause disease such as serum sickness. Such a situation can be improved by using the antibody of the present invention. In this embodiment, such antibodies are administered in combination with a therapeutic or diagnostic agent. The addition of the antibody prevents the recipient from recognizing the drug and thus prevents the recipient from mounting an immune response to the drug. The absence of such an immune response results in the patient's ability to accept additional administration of a therapeutic or diagnostic agent.

F. Use of Intercellular Adhesion Molecule-1 (ICAM-1) ICAM-1 is a binding partner of LFA-1. Thus, ICAM-1 or a functional derivative thereof can be used interchangeably with antibodies capable of binding LFA-1 in the treatment of disease. That is, in a solubilized form, such molecules may be inflammatory,
It can be used to suppress organ rejection, transplant rejection and the like. ICAM−
One or a functional derivative thereof can be used in the same manner as an anti-ICAM antibody to reduce the immunogenicity of a therapeutic or diagnostic agent.

ICAM-1, its functional derivatives, and its antagonists can be used to block the metastasis or growth of tumor cells that express ICAM-1 or LFA-1 on their surface. Various methods can be used to achieve such a purpose. For example, hematopoietic cell penetration requires LFA-1-ICAM-1 binding. Thus, antagonists of such binding inhibit the above penetration and block the metastasis of leukocyte-derived tumor cells. A toxin-derived molecule that can bind to a member of the ICAM-1 or LFA-1 family of molecules can also be administered to the patient. When such a toxin-derived molecule binds to a member of the ICAM-1 or LFA-1 family of molecules, the presence of the toxin kills tumor cells and thereby suppresses tumor growth.

G. Use of Non-Immunoglobulin Antagonists for ICAM-1-Dependent Adhesion ICAM-1-dependent adhesion can be suppressed by non-immunoglobulin antagonists that can bind to ICAM-1 or LFA-1. One example of a non-immunoglobulin antagonist of ICAM-1 is LFA-1. An example of a non-immunoglobulin antagonist that binds to LFA-1 is ICAM-1. By using the aforementioned assays, additional non-immunoglobulin antagonists can be identified and purified. Non-immunoglobulin antagonists of ICAM-1-dependent adhesion can be used for the same purpose as antibodies to LFA-1 or antibodies to ICAM-1.

H. Administration of the Compositions of the Invention The therapeutic effect of ICAM-1 is obtained by administering to a patient all ICAM-1 or any therapeutically active peptide fragment thereof.

ICAM-1 and its functional derivatives can be obtained synthetically using recombinant DNA or by proteolysis. I c
The therapeutic benefit of AM-1 can be enhanced by the use of functional derivatives of ICAM-1 having additional amino acid residues to improve binding to the carrier or to improve ICAM-1 activity. The scope of the present invention is further intended to include functional derivatives of ICAM-1 that lack certain amino acid residues or contain other amino acid residues, as long as such derivatives exhibit the ability to affect cell adhesion. .

Antibodies and ICAM-1 molecules of the present invention are said to be "substantially free of natural impurities" if the preparations containing them are substantially free of substances normally found in nature with these products.

The invention also extends to antibodies capable of binding to ICAM-1 and biologically active fragments thereof (polyclonal or monoclonal). Such antibodies can be prepared by animal, tissue culture or recombinant DNA means.

To administer an antibody or a fragment thereof capable of binding to ICAM-1 to a patient, or to receive and administer ICAM-1 (or a fragment, variant, or derivative) to a patient, the dosage should be the age of the patient. , Weight, height, gender, general medical conditions, medical history and other factors. Generally, it is desirable to administer to the patient an antibody dosage in the range of about 1 Pg / kg to 10 mg / kg (patient weight), although lower or higher doses can be administered. When administering the ICAM-1 molecule or a functional derivative thereof to a patient,
It is preferred to administer such molecules at a dose in the range of 1 Pg / kg to 10 mg / kg (based on the weight of the patient), although lower or higher doses can be used. As described below, the effective dose can be reduced when the anti-ICAM-1 antibody is co-administered with anti-LFA-1 administration. In the context of the present invention, one compound can be co-administered to a patient when administration of the second compound and the two compounds results in conditions such that both compounds can be detected simultaneously in the patient's serum.

Both antibodies capable of binding to ICAM-1 and ICAM-1 itself can be administered to patients intravenously, intramuscularly, subcutaneously, enterally or parenterally. When administering the antibody or ICAM-1 by injection, the administration can be by continuous infusion or by single or multiple boluses.

The anti-inflammatory agent of the present invention is to be administered to a recipient subject in an amount sufficient to suppress inflammation. The amount may be sufficient to "suppress" inflammation when the dose, route of administration, etc. of the agent is sufficient to attenuate or prevent inflammation.

The anti-inflammatory agent of the present invention can be administered either before onset of inflammation (to suppress expected inflammation) or after onset of inflammation.

Anti-ICAM-1 antibodies or fragments thereof can be administered alone or in combination with one or more additional immunosuppressive agents (particularly for recipients of organ or tissue transplants). Administration of such compounds can be for either "prophylactic" or "therapeutic" purposes. When administered prophylactically, these immunosuppressive agents are administered prior to an inflammatory response or symptom (eg, prior to organ or tissue transplantation, during or immediately after transplantation, prior to organ rejection symptoms). Prophylactic administration of these compounds helps to prevent or reduce any subsequent inflammatory response, such as rejection of transplanted organs or tissues. When administered therapeutically, these immunosuppressive compounds are administered at (or shortly after) the onset of actual symptoms of inflammation, such as, for example, organ or tissue rejection. Therapeutic administration of these compounds helps to reduce any actual inflammation (eg, rejection of transplanted organs or tissues). Thus, the anti-inflammatory agents of the invention can be administered either before the onset of inflammation (to suppress the expected inflammation) or after the onset of inflammation.

A composition is said to be "pharmaceutically acceptable" if its administration is acceptable to the receiving patient. Such an agent is said to be administered in a "therapeutically effective amount" if the dose is physiologically significant. An agent is financially significant if its presence results in a detectable change in the physiology of the receiving patient.

The antibodies and ICAM-1 molecules of the present invention can be formulated by known methods of preparing pharmaceutically useful compositions, whereby these substances or functional derivatives thereof can be combined as a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and preparations thereof (including, for example, other human proteins, such as human serum albumin) are described, for example, in "Remington's Pharmaceutical Sciences (16th Edition, Dso
l, A. Ed., Mark Easton (PA), (1980) ". To prepare a pharmaceutically acceptable composition suitable for effective administration, such compositions should be in an appropriate amount. An effective amount of an anti-ICAM-1 antibody with a carrier vehicle of
Contains the ICAM-1 molecule, or a functional derivative thereof.

The duration of activity can be controlled by additional pharmaceutical methods. Controlled release formulation is anti-ICAM-1 antibody or ICAM
-1, or their functional derivatives, by using polymers that complex or absorb them. Controlled transmission is achieved by appropriate macromolecules (eg,
Polyester, polyamino acid, polyvinylpyrrolidone,
Ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of the macromolecule and the method of incorporation to control release. Another possible way to control the duration of activity with controlled release formulations is anti-IC
Incorporating the AM-1 antibody or ICAM-1 molecule, or a functional derivative thereof, into particles of a polymeric material such as a polyester, polyamino acid, hydrogel, polylactonic acid or ethylene-vinyl acetate copolymer. Instead of mixing these drugs into the polymer particles, these drugs are added to macrocapsules prepared by, for example, a coacervation method or an interfacial polymerization method, for example, hydroxymethyl cellulose or gelatin microcapsules and (methyl methacrylate) microcapsules. Or colloidal drug delivery systems such as liposomes,
Albumin microspheres, microemulsions,
It is also possible to enclose them in nanoparticles, nanocapsules or macroemulsions. Such a method is also called “Remington's Pharmaceutical Sciences (1980)”
It is described in.

EXAMPLES While the present invention has been described in general terms, the following examples will make the invention easier to understand. These examples are for illustrative purposes only and do not limit the invention.

Example 1 Culture of Animal Cells In general, each EBV-transformed and hybridoma cell of the invention was RMP11640 supplemented with 20 mM L-glutamine, 50 μg / m gentamicin, and 10% fetal bovine serum.
Stored in medium. The cells were cultured at 37 ° C. in an atmosphere of 5% CO ₂ and 95% humidity.

To establish Epstein-Barr virus (EBV) transformants, 20% fetal calf serum (FCS) and 50 μg
10 ^{6 in} RPM11640 medium with gentamicin / m
T cell exsanguinated peripheral mononuclear cells / m were incubated with EBV-containing supernatant of B95-8 cells for 16 hours [Thorles-Lawso
n, DA, et al., J. Exper . Med . 146 : 495 (1977)]. Aliquots of 0.2 m cells were placed in 10 microtiter wells. The medium was RPMI 1640 medium (20% fetal bovine serum and 50 μm /
Gintamycin) was added until cell growth was seen. Cells grew in most wells and spread in the same medium. Phytohemagglutinin (PHA) blasts diluted 1: 800 PH
10 ⁶ cells in RPMI 1640 medium (with 20% fetal calf serum) containing AP (Difco Laboratories, Detolite, MI)
Established at / m. The PHA system is interleukin 2 (IL-
2) Spread in conditioned medium and weakly pulsed by PHA [Cantrel
J. Exper . Med . 158 : 1895 (1983)]. The above procedure is described in “Springer, T. et al., J. Exper . Med . 160 : 1901-1918 (19
84) ", the description of which is incorporated herein by reference. The cells obtained by the above procedure were then used for anti-LFA
Screening with the -1 antibody determined whether they expressed the LFA-1 antigen. One such antibody is “Sanc
Hey Madrid, F. et al., J. Exper . Med . 158: 1785 (1983) ".

Example 2 Cell Aggregation and Adhesion Assay To assess the degree of cell adhesion, an aggregation assay was used. The cell line used for such an assay contains 5 mM Hepes buffer (Sigma Chemical, St. Louis).
Washed twice with PRMI1640 medium and resuspended to a concentration of 2 × 10 ⁶ cells / m. Flat bottom 96-well microtiter plate (No. 3596; Coastar, Cambridge, Mass.) With 50 μl of the appropriate monoclonal antibody supernatant, 50 μl of complete medium with or without purified monoclonal antibody, 50 μl of 20
Cells were added at a concentration of 2 × 10 ⁶ cells / m in complete medium containing 0 ng / m phorbol ester phorbol myristate acetate (PMA) and 100 μl of complete medium. This is 50ng / m
PMA and a final concentration of 2 × 10 ⁵ cells / well were given. Cells were spontaneously settled and the degree of aggregation was counted at each time point. Frequencies ranged from 0-5 ⁺ . Where 0 indicates that there is essentially no cells in the confluence; ¹⁺ indicates that less than 10% of the cells are in the aggregate; ²⁺ indicates that less than 50% of the cells are aggregated:
3 ⁺ indicates that there in the cell is small gradual dense to 100%; 4 ⁺ indicates that the cells of up to 100% are aggregated into large dense; 5 ⁺ extremely 100% of the cells is greater Indicates that it was in a dense aggregate. In order to obtain a more quantitative assessment of cell adhesion, reagents and cells were 5m in the same order as above.
Added to polystyrene tubes. The tubes were placed at 37 ° C. in a platform on a gliatory shaker. After 1 hour at about 200 rpm, 10μ of the cell suspension was placed in a hemocytometer and the number of free cells was quantified. % Aggregation was determined by the following equation:

The number of cells included in the above equation is the number of cells per m in control tubes containing only unincubated cells and complete medium. The number of free cells in the above equation equals the number of non-aggregated cells per m from the test tube. The above procedure is described in “Rothlein, R., et al., J. Exper .
63 : 1132-1149 (1986) ".

Example 3 LFA-1-Dependent Cell Aggregation The quantitative agglutination assay described in Example 2 was performed using the Epstein-Barr virus transformed cell line JY.
PMA was added to the medium in the microtiter plate and cell aggregation was observed. The time-lapse video recorder showed that the JY cells at the bottom of the microtiter well were moving, showing active membrane wave motion and pseudopod movement. Contact between pseudopodia of adjacent cells often resulted in cell-cell adhesion. If sticking persisted, the cell contact area moved to the uropod. Contact could be maintained despite vigorous cell movement and cell pulling in opposite directions. The major difference between PMA-treated and untreated cells appeared in the stability of the contact once formed. In PMA, cell confluence appeared and there was granular growth as additional cells sticking around.

As a second means of measuring adhesion, the quantitative assay described in Example 2 was used. Cell suspension for 2 hours, 20
The cells were shaken at 0 rpm, transferred to a hemocytometer, and the cells not contained in the aggregate were counted. In the absence of PMA, 42% (SD
= 20%, N = 6) in the aggregates after 2 hours, while 87% (SD = 8% SD) of the JY cells incubated under the same conditions with 50 μg / m PHA , N = 6). Kinetic studies of aggregation showed that PMA promoted aggregation rate and strength at all test times (FIG. 3).

Example 4 Inhibition of Cell Aggregation Using Anti-LFA-1 Monoclonal Antibodies To test the effect of anti-LFA-1 monoclonal antibodies on PMA-induced cell aggregation, such antibodies were tested according to the quantitative agglutination assay of Example 2. The incubated cells were added. This monoclonal antibody was found to suppress the formation of cell aggregates in both the presence and absence of PMA. Both the F (ab ') ₂ and Fab' fragments of the monoclonal antibody against the alpha chain of LFA-1 were able to inhibit cell aggregation. On the other hand, essentially 100%
Cells form aggregates in the absence of anti-LFA-1 antibody,
When antibodies were added, less than 20% of the cells were found in the aggregates. The results of this experiment were disclosed by Rothlein, R. et al. ( J. Exper. Med. 163 : 1132-1149 (1986)).

Example 5 Cell aggregation requires the LFA-1 receptor: EBV transformed lymphoblast cells were prepared from patients by the method described in Example 1. The cells were screened for a monoclonal antibody that can recognize LFA-1,
The cells were found to be LFA-1 deficient.

The quantitative agglutination assay described in Example 2 was performed using the above LFA-1.
This was performed using defective cells. The cells did not spontaneously aggregate in the presence of PMA.

Example 6 Discovery of ICAM-1 The LFA-1-deficient cells of Example 5 were labeled with carboxyl fluorescein diacetate [Patarroyo, M. et al., Cel .
l . Immunol. 63 : 237-248 (1981)]. The labeled cells were mixed with the same or JY cells at a ratio of 1:10, and the ratio of fluorescein-labeled cells in the aggregate was determined as “Rothlein, R. et al .
63 : 1132-1149 (1986). "
-1 deficient cells were found to be able to coaggregate with LFA-1 expressing cells (FIG. 4).

To determine whether LFA-1 is only important for aggregate formation or its maintenance, antibodies capable of binding LFA-1 were added to the pre-formed aggregates described above. The addition of the antibody was found to strongly disrupt the previously formed aggregates. Time course video recorders showed that the addition of the monoclonal antibody to the preformed aggregates began to divide within 2 hours (Table 1). Following the addition of the monoclonal antibody to LFA-1, pseudopodia movement and shape salinity of the individual cells during aggregation continued unchanged. Individual cells gradually dissociated from the periphery of the aggregate, and by 8 hours the cells were almost dispersed. LFA according to video time lapse
The disruption of pre-formed aggregates with the -1 monoclonal antibody was equivalent to a reverse course of aggregation in the absence of the LFA-1 monoclonal antibody.

Aggregation in quantitative microtiter plates was counted observable. Anti-LFA-1 present throughout assay time
In, the aggregation was ¹⁺ or less. ^a Aggregation amount 2 hours immediately before addition of monoclonal antibody ^b TS1 / 18 + TS1 / 22 ^c TS1 / 18 ^d TS1 / 22 Example 7 Necessity of divalent ion for LFA-1-dependent aggregation LFA- between cytotoxic T cells and target 1-dependent adhesion requires the presence of magnesium [Martz, E., J. Cell.
Bioll. 84 : 584, 598 (1980)]. PMA-induced JY cell aggregation was tested for divalent cation dependence. JY cells did not aggregate in media without calcium or magnesium ions (using the assay of Example 2). Addition of divalent magnesium caused aggregation at a concentration as low as 0.3 mM. Addition of calcium ions alone had little effect. However, causium ions were found to enhance the ability to assist PMA-induced aggregation of magnesium ions. It was found that when 1.25 mM calcium ion was added to the medium, aggregation was assisted by a magnesium ion concentration as low as 0.02 mM. These data show the cellular LFA-1.
It shows that dependent aggregation requires magnesium ions and that calcium ions increase aggregation with magnesium ions, albeit poorly.

Example 8 Isolation of hybridoma cells capable of expressing anti-ICAM-1 monoclonal antibody Monoclonal antibodies capable of binding to ICAM-1 were identified as "Rothle
in, R., et al ., J. Immunol . 137 : 1270-1274 (1986) ", which is incorporated herein by reference. Three BALB / C mice were EBV-transformed peripheral blood from an LFA-1 deficient patient. Intraperitoneal immunization with mononuclear cells (Spr
inger, TA, et al., J. Exper. Med. 160: 1901 (1984). 1m R
Approximately 10 ⁷ cells in PML1640 medium was used for each immunization. Immunization was performed on days 45, 29 and 4 before spleen cells were removed from mice.
Day to produce the desired hybridoma cell line. Three days before removal of spleen cells, mice received an additional 10 ⁷ cells in 0.15m medium (iv).

Isolated spleen cells from the above animals were compared to P3 × 73Ag8.653 myeloma cells in a 4: 1 ratio according to “Galfre, G. et al., Nature 266 : 55.
0 (1977) ". Each aliquot of the resulting hybridoma cells was placed in a 96-well microtiter plate. Each hybridoma supernatant was screened for aggregation inhibition and one inhibitory hybridoma (all 600 or more From among the well tests) and subcloned by limiting dilution, designated RP1 / 1.1.1. (Hereinafter designated "PR1 / 1").

Monoclonal resistance RP1 / 1 is an LFA-1 expressing cell line JY
Was found to consistently suppress PMA-stimulated aggregation of liposomes. R
The P1 / 1 monoclonal antibody inhibited aggregation less than or equivalently to monoclonal antibodies to several LFA-1 α or β subunits. In contrast, the control monoclonal antibody to HLA, which is expressed in large amounts on JY cells, did not inhibit aggregation. The antigen bound to monoclonal antibody RP1 / 1 is defined as intercellular adhesion molecule-1 (ICAM-1).

Example 9 Use of Anti-ICAM-1 Monoclonal Antibody to Characterize ICAM-1 To limit the properties of ICAM-1, in particular, ICAM-1 was LF
To determine if it was different from A-1, cell proteins were immunoprecipitated using the monoclonal antibody RP1 / 1. The immunoprecipitation was performed according to the method of Rothlein, R. et al . [ J. Immunol . 137: 1270-1274 (1986)] JY cells were renewed with 1 mM phenylmethylsulfonyl fluoride, 0.2 units.
/ m with trypsin inhibitor aprotinin
% Triton X-100, 0.14 mM NaCl, 10 mM Tris, pH 8.
0 (lysis buffer) at 5 × 10 ⁷ cells / m for 20 minutes, 4
Dissolved at ° C. Lysate is centrifuged at 10,000 xg for 10 minutes and CNBr
50% suspension of activated glycine-suppressed Sepharose Cl-4B 50
m for 1 hour at 4 ° C. Monoclonal antibody (1 mg / ml) conjugated with 1 m lysate to Sepharose Cl-4B
m) at 20 ° C. overnight at 4 ° C. [Springer, TA et al., J. Exper. Med. 160: 1901 (198
Four)〕. Sepharose-conjugated monoclonal antibody is called “Marc
h, S. et al., prepared using CNBr activation of Sepharose Cl-4B in carbonate buffer [ Anal.
chem . 60 : 149 (1974)]. The washed immunoprecipitate was subjected to SDS-PA
"Morrissey, JHAnal. Biochem. 117: 3
07) 1981) ".

SDS sample buffer (HD, MK, etc., J. Biol . Chem. 2
58 : 636 (1983)], each sample was divided into halves, and electrophoresis (SDS-8%) was performed under reducing (Fig. 5A) or non-reducing (Fig. 5B) conditions. PAGE). The bands with molecular weights of 50 Kd and 25 Kd corresponded to the long and short chains of immunoglobulins from the monoclonal antibody Sepharose (FIG. 5A, lane 3). 25-50Kd
Other bands of varying molecular weight range were also observed, but were not found in sediments from hairy leukemia cells, which gave only the 90 Kd molecular weight band. 177Kd of LFA-1
α and 95Kdβ subunits are reduced (5A
It permeated differently from ICAM-1 under both conditions (Figure, lane 2) and non-reducing (FIG. 5B, lane 2).

Monoclonal antibody R against PHA-lymphoblast aggregation
To determine the effect of P1 / 1, the quantitative agglutination assay described in Example 2 was used. In other words, T cell blast cells were
Stimulated for one day, washed extensively, and then cultured in the presence of IL-2 conditioned medium for 6 days. PHA was incorporated in this 6-day culture and did not contribute to the aggregation assay. In three assays with different T cell blast preparations, the ICAM-1 monoclonal antibody consistently inhibited aggregation (second
table).

^a Aggregation of PHA-induced lymphoblasts stimulated with 50 ng / m PHA was indirectly quantified by microscopically counting the number of non-aggregated cells as described in Example 2. ^b % inhibited ^c- aggregation for cells treated with PMA and X63 monoclonal antibody was measured one hour after stimulatory addition of monoclonal antibody and PMA. ^d Negative numbers indicate% promotion of aggregation. ^e- aggregation was measured one hour after the stimulatory addition of the monoclonal antibody and PMA. Cells were pelleted at 200 × G for 1 minute, incubated at 37 ° C. for 15 minutes, gently resuspended, and shaken at 100 rpm for 45 minutes. ^f cells were pretreated with PHA at 37 ° C for 4 hours. After the addition of the monoclonal antibody, each tube was incubated at 37 ° C. for 20 minutes and shaken at 75 rpm for 100 minutes.

The LFA-1 monoclonal antibody is consistently more inhibitory than the ICAM-1 monoclonal antibody, while HLA-A,
B and LFA-3 monoclonal antibodies had no effect. These results indicate that of the monoclonal antibodies tested, only those that can bind to LFA-1 or ICAM-1 can inhibit cell adhesion.

Example 10 Preparation of Monoclonal Antibody Against ICAM-1 Immunization Balb / c mice were immunized intraperitoneally (ip) with 0.5 × 2 × 10 ⁷ JY cells in RPMI medium 103 and 24 days before fusion. On days 4 and 3 before fusion, mice were placed in 0.5m RPMI medium.
PMA differentiated U937 cells were immunized ip.

Differentiation of U937 cells U937 cells (ATCC CRL-1593) were isolated from 10% fetal calf serum, 1% glutamine and 50 ng / m phorbol-
Differentiation was achieved by incubation in sterile polypropylene containers at 5 × 10 ⁵ / m in RPMI containing dietamycin (complete medium) containing 12-myristate acetate (PMA). On the third day of this incubation, 1/2 volume of the medium was removed and replaced with fresh complete medium containing PMH. 4
At day, cells were removed, washed and prepared for immunization.

Fusion Spleen cells from immunized mice were P3 × 63 according to Galfre et al.
Ag8.653 myeloma cells were fused at a 4: 1 ratio ( Nature
266: 550 (1977)]. After fusion, it was placed in 10 ⁵ spleen cells / well cells in 96-well flat-bottom microtiter plates.

Selection of anti-ICAM-1 positive cells One week later, 50 μl of supernatant was screened in the quantitative agglutination assay of Example 2 using both JY and SKW-3 as agglutination cell lines. Suppresses JY cell aggregation but SKW-
3. Cells from the supernatant that did not inhibit aggregation were selected and cloned twice using limited dilution.

This experiment allowed the identification and cloning of three hybridoma lines producing anti-ICAM-1 monoclonal antibodies. Antibodies produced by these hybridoma lines were respectively IgG _2a, IgG _2b and IgM. I
gG _2a anti-ICAM-1 antibody hybridoma cell lines that produce was displayed R6'5'D6'E9'B2. The antibody produced by this preferred hybridoma cell line is R6'5'D6'E9'B2
(Hereinafter referred to as "R6-5-D6").

Example 11 ICAM-1 Expression and Normalization To measure ICAM-1 expression, a radioimmunoassay was performed. In this assay, purified RP1 / 1 was iodinated with iodogen to a specific activity of 10 μCi / μg. Endothelial cells were grown in 96-well plates and treated as described in each experiment. Each plate was cooled to 4 ° C by placing it in a cold room for 0.5-1 hour instead of directly on ice. Wash each monolayer three times with cold complete medium and then at 4 ° C.
Incubated with ¹²⁵ I RP1 / 1 for 30 minutes. The monolayer was then washed three times with complete medium. Binding ¹²⁵ I to 0.1N NaOH
Released and counted. The specific activity of ¹²⁵ I RP1 / 1 was adjusted using unrecognized RP1 / 1 to obtain a linear signal over the antigen density range used in this study. Non-specific binding
Subtractive specific binding was obtained from total binding measured in the presence of a 1,000-fold excess of unlabeled RP1 / 1.

ICAM expression, measured using the radioimmunoassay described above, was determined on human nasal vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) by IL-1, TNF-, LPS and I.
Increased by NF gamma (Table 3). Saphenous vein endothelial cells were used in this study to confirm results from navel vein endothelial cells in cultured vena cava endothelial cells from adult tissues. The basal expression of ICAM-1 is twice as high on saphenous vein endothelial cells as on nasal vein endothelial cells. Recombinant IL-1 alpha, IL-1 beta, and TNF in nasal vein endothelial cells
Exposure increases ICAM expression by 10-20 fold. IL-1 alpha, TNF and LPS are the most potent inducers,
IL-1 is less potent on a weight basis and at saturating concentrations of response (Table 3). At 100 ng / m, IL-1 beta is I
CAM-1 expression was increased 9-fold on HUVEC and 7.3-fold on HSVEC, with a half-maximal increase occurring at 15 ng / m. 50 ng / m rTNF
Increased ICAM-1 expression 16-fold on HUVEC and 11-fold on HSVEC, with a half-maximal effect of 0.5 ng / m. Interferon gamma is HUV at 10,000 U / m in ICAM-1 expression
It showed a significant increase of 5.2 fold on EC or 3.5 fold on HSVEC. The effect of LPS at 10 μg / m was similar in strength and effect of rTNF. The combination of these mediator pairs is ICAM
-1 resulted in an additional effect or slightly less than the additional effect (Table 3). rTMF and rIL
Cross-titration with -1beta or rIFN gamma showed no synergy in effect at suboptimal or best concentrations.

Since LPS increased ICAM-1 expression on endothelial cells on a basis sometimes found in media, basal ICAM-1
The possibility that expression could be based on LPS was tested. When several serum batches were tested, 25 low endotoxin serum was found.
% Low ICAM-1 basal expression. All results presented here were for endothelial cells grown in low endotoxin serum. However,
Addition of the LPS neutralizing antibiotic polymyxin B at 10 μg / m slightly reduced ICAM-1 expression to an additional 25% (Table 3), ICAM-1 expression upon treatment with IL-1 or TNF The increase was not effected by the presence of 10 μg / m polymyxin B, which was consistent with low endotoxin values in these preparations (Table 3).

Upregulation of ICAM-1 expression on HVEC and HSVEC-HUVEC or HSVEC is shown in 96-well plates:
At 3 seeds were grown from the confluent monolayer and grown in confluence. The cells were then treated with each substance or medium for 16 hours and RIA was performed methodically. All figures were repeated four times.

Example 12 Interleukin-1 and gamma-interferon-induced kinetics of ICAM-1 The kinetics of the effects of interleukin-1 and gamma-interferon on ICAM-1 expression on dermal fibroblasts were measured by ¹²⁵ I goats such as Dustin, ML. Measured using an anti-mouse IgG binding assay. [ J. Immunol , 137 : 245-254 (198
6); the reference is incorporated herein by reference. To perform this binding assay, human skin fibroblasts were grown in 96-well microtiter plates at 2-8 × 10 ⁴ cells / well (0.32 cm ² ). Cells were washed twice with RPMI 1640 medium supplemented as described in Example 1.
Cells were further diluted with Hank's Balanced Salt Solution (HBSS), 10 mM HEPE
S, 0.05% NaN ₃ and 10% heat-inactivated fetal calf serum
Washed twice. Washing with this binding buffer was performed at 4 ° C. 50 μl of the above binding buffer and
Suitable hybridoma supernatant 50m with X63 and W6 / 32
Was added as negative and positive controls, respectively. After incubation for 30 minutes at 4 ° C., the wells were washed twice with binding buffer and the second antibody ¹²⁵ I-goat anti-mouse IgG was added at 50 nCi in 100 μ. ¹²⁵ I-goat anti-mouse antibody was obtained using Fraker, Iodogen (Pierce).
It was prepared according to a method such as PJ ( Biochem. Biophys. Res.
Commun . 80 : 849 (1978)]. After 30 minutes at 4 ° C, add 2 wells
Wash twice with binding buffer 200 μl and remove cell layer 100 μl
Of 0.1N NaOH. This treatment and the 100μ wash were counted in a Beckman 5500 gamma counter. The specific count of binding per minute was [cpm by monoclonal antibody]-[cpm by X63]
Calculated as All steps, including induction with specific reagents, were performed in four replicates.

The effect of interleukin 1 with a half-life of 2 hours on ICAM-1 induction was more rapid than that of gamma-interferon with a half-life of 3.75 hours (FIG. 6).
The time to return to quiescent levels of ICAM appeared to depend on cell cycle or cell proliferation. In quiescent cells, the interleukin 1 and gamma-interferon effects are stable for 2-3 days, while in log phase cultures, ICAM-1 expression is close to baseline 2 days after removal of these inducers.

IC with recombinant mouse and human interleukin 1
FIG. 7 shows dose response curves of AM-1 induction and ICAM-1 induction by recombinant gamma interferon. Gamma interferon and interleukin 1 were found to have a similar concentration dependence with almost the same effect at 1 ng / m. Human and mouse recombinant interleukin 1 have similar curves, but ICAM-
1 shows a significantly lower effect than the human interleukin 1 preparation in inducing expression.

Cycloheximide (an inhibitor of protein synthesis) and actinomycin D (an inhibitor of mRNA synthesis) counteract the effects of interleukin-1 and gamma-interferon on ICAM-1 expression on fibroblasts (Table 4). Furthermore, only tunicamycin (an inhibitor of N-linked glycosylation) suppressed the interleukin 1 effect by 43%. These results indicate that protein and
It shows that mRNA synthesis is required for interleukin-1 and gamma-interferon-induced increase in ICAM-1 expression, whereas N-linked glycosylation is not.

^a Human fibroblasts were grown to a density of 8 × 10 ⁴ cells / 0.32 cm ² well. Processing was performed at a final concentration of 50μ containing each reagent. Cycloheximide, actinomycin D and tunicamycin were added at the same time as the cytokines at 20 μg / m, 10 μM and 2 μg / m, respectively. All figures are means ± SD of quadruplicate wells.

Example 13 Tissue distribution of ICAM-1 Histochemical tests were performed on frozen tissues of human organs to measure the distribution of ICAM-1 in the thoracic line, lymph node, intestine, skin, kidney and liver. To perform such an analysis, a frozen human tissue section (4 μm thick) of normal human tissue was fixed in acetone for 10 minutes with a monoclonal antibody, RR1 / 1, using Cerf-Bensusss.
Adipine-biotin complex method disclosed by An, N. et al. (Vector Laboratories, Burlingame, C.
Staining was performed by the immunoperoxidase method using A) [ J. Immunol. 130 : 2615 (1983)]. After incubation with the antibody, sections were sequentially incubated with biotinylated horse anti-mouse IgG and avidin-biotinylated peroxidase complex. Each section was ultimately 3-amino-
9-ethyl-carbazole (Aldrich Chemical Company,
A color reaction was performed by immersion in a solution containing (Milwaukee, WI). Each section was then fixed in 4% formaldehyde for 5 minutes and counterstained with hematoxylin (counters
tain). Controls included sections incubated with an irrelevant monoclonal antibody instead of the RRI / 1 antibody.

ICAM-1 was found to have almost the same distribution as the major histocompatibility complex (MHC) class II antigen. Most of the blood vessels (both large and small) in all tissues showed endothelial cell staining with the ICAM-1 antibody. Ductal endothelial staining is stronger in the intervesicular (paracortical) areas in lymph nodes, tonsils and Paier plaques compared to blood vessels in the kidney, liver and normal skin. In the liver, staining was mostly limited to sinusoidal capillary lining cells, and most lining hepatocytes and endothelial cells of portal veins and arteries were not stained.

In the thoracic chord, large cells showed diffuse staining and tree staining. In the cortex, the stained image was nest-like and mainly tree-like. Thymocytes were not stained. In peripheral lymphocyte tissues, germinal center cells of secondary lymphoid follicles were strongly stained. In some lymphoid follicles, the stained image was mostly dendritic, with no recognizable staining of lymphocytes. A slight staining of cells in the mantle region was also observed. In addition, dendritic cells with cytoplasmic enlargement (interdigital reticular cells) and a small number of lymphocytes within the vesicles or in the paracortical region were stained with ICAM-1 binding monoclonal antibodies.

Cell-like macrophages were stained in thin-film propria of lymph nodes and small intestine. Fibroblast-like cells (spindle-shaped cells) that had diffused into most of the stroma of the organs examined were stained with ICAM-1 binding antibodies.
No staining was observed in Langerhans / adult cells in the hull. No staining was observed even in smooth muscle tissue.

Epidermal cell staining was consistently seen in the tonsil mucosa. Hepatocytes, hepatic tract hulls, intestinal husk cells, and tubular husk cells in the kidney were not stained in most situations, but sections of normal kidney tissue from resected sections with renal cell carcinoma were ICAM- One showed staining of many adjacent tubular cells. These tubular envelope cells also stained with anti-HLA-DR binding antibodies.

In summary, ICAM-1 is expressed on non-hematopoietic cells, such as tubular envelope cells, and on hematopoietic cells, such as tissue macrophages and mitogen-stimulated T lymphoblasts. ICAM−
1 was found to be expressed in small amounts in peripheral blood lymphocytes.

Example 14 Purification of ICAM-1 by Monoclonal Antibody Affinity Chromatography General Purification Procedure ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography.
The monoclonal antibody RP1 / 1 reactive with ICAM-1 was first purified and bound to an inert column matrix. This antibody is described in "Rothlein, R. et al. , J. Immunol. 137 : 1270-1274 .
(1986) "and" Dustin, ML, et al. , J. Immunol. 137 : 2 .
45 (1986) ". ICAM-1 was transfected from cell membranes into cells at near neutral pH in the nonionic detergent Triton X-100.
And solubilized. The cell lysate containing solubilized ICAM-1 is passed through a pre-column designed to remove substances that bind non-specifically to the column matrix material, and then passed through a monoclonal antibody column matrix to bind ICAM-1 to the antibody. I let it. Antibody column
Washing was performed with a series of detergent washing buffers in which the pH was raised to pH 11.0. During these washes, ICAM-1 remained bound to the antibody matrix and unbound and weakly bound impurities were removed. Then the combined ICAM-1 is
The column was specifically eluted by applying a detergent buffer at pH 12.5.

Purification of monoclonal antibody RR1 / 1 and Sepharose CL
Covalent binding to -4B The anti-ICAM-1 monoclonal antibody RP1 / 1 was purified from ascites fluid or hybridoma culture supernatant of hybridoma-containing mice by standard ammonium sulfate precipitation and protein A affinity chromatography (Ey et al.
Immunochem. 15: 429 (1978)]. Purified IgG or rat I
gG (Sigma Chemical Co., St. Louis, MO) transferred to Sepharose CL-4B (Pharmacia, Sweden) March
Covalent binding was carried out using a modification of the method described in [Anal. Biochem. 60: 149 (1974)]. In short, Sepharose CL-4B
Was washed with distilled water, and 40 mg / m in 5M K ₂ HPO ₄ (pH about 12)
Activated with CNBr for 5 min, then washed long with 0.1 mM HCl at 4 ° C. Filtered and purified antibody with an equal volume of Sepharose activated (0.1M NaHCP _3, 0.1M NaCl in 2 to 10 mg / m) was resuspended at. The suspension was incubated at 4 ° C. for 18 hours by gentle end-to-end rotation. The supernatant was then monitored for unbound antibody by absorbance at 280 nm,
Glycine was added to the remaining reactive site of activated Sepharose to 0.05.
Saturated by adding to M. Coupling efficiency is typically 90
% Or more.

Detergent solubilization of membranes prepared from human spleen All procedures were performed at 4 ° C. Frozen human spleen (200g fragment) from a patient with hairy cell leukemia on ice
1 mM phenylmethylsulfonyl fluoride (PMS
F), 200m Tris-brine (50mM Tris: 0.14M) containing 0.2U / m aprotinin and 5mM iodoacetamide.
NaCl, pH 7.4 at 4 ° C.). The tissue was cut into small pieces and homogenized at 4 ° C. with a Temer strong homogenizer. The capacity is 300m with Tris-brine, and Tris-brine
Add 100m of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) to a final concentration of 2.5% Tween 4
Got 0.

To prepare the membrane, the homogenate is extracted using a three-stroke Dounce or more preferably using a Teflon potter elb jam homogenizer and then 1000xg
For 15 minutes. The supernatant was retained and the pellet was re-extracted with 250m of 2.5% Tween 40 in Tris-brine. 1000x
After centrifugation at g for 15 minutes, the supernatants from both extracts are combined and
The membrane was pelleted by centrifugation at 0,000 × g for 1 hour. 200 membranes
Washed by resuspension in 1 ml of Tris-brine and centrifuged at 150,000 xg for 1 hour. 200m membrane pellet
And then homogenized with a motor-driven homogenizer and Teflon pestol until the suspension was thick. The volume was then brought to 900 m with Tris-brine and N-lauroyl sarcosine was added to a final concentration of 1%. After stirring at 4 ° C. for 30 minutes, insoluble substances in the detergent lysate were removed by centrifugation at 150,000 × g for 1 hour. Triton X-100 was added to the supernatant to a final concentration of 2% and the lysate was stirred at 4 ° C for 1 hour.

JYB- detergent solubilization of lymphoblastoid cells EBV transformed B- lymphoblastoid cell line JY an in RPMI 1640 containing 10% fetal calf serum (FCS) and 10mM HEPES 0.8~1.0 × 1.0 ⁶ cells / m Were grown to a density of ICAM−
To increase cell surface expression of 1, phorbol 12-myristate 13-acetate (PMA) was added at 25 ng / m for 8-12 hours before harvesting the cells. Sodium vanadate (50 μM) was also added to the culture during this time. 50 cells
Pelleted by centrifugation at 0xg for 10 minutes and washed twice by resuspension and centrifugation in Hans Balanced Salt Solution (HBSS). 50 cells (about 5 g per 5 cultures)
m lysis buffer (0.14 M NaCl, 50 mM Tris, pH 8.
0, 1% Triton X-100, 0.2 μ / m aprotinin, 1 mM
PMSF, 50 μM sodium vanadate) by stirring at 4 ° C. for 30 minutes. Undissolved nuclei and insoluble pieces are centrifuged at 10,000 × g for 15 minutes, then 150,000 × g
The supernatant was removed by centrifugation for 1 hour at room temperature and filtration of the supernatant through Whatman 3 mm filter paper.

Affinity chromatography of ICAM-1 for structural studies As a large-scale purification of ICAM-1 used for structural studies, 10
m RP1 / 1-Sepharose CL-4B column (bound with 2.5 mg antibody / gel m) and CNBr-activated, glycine stabilized Sepharose CL-4B and rat IgG bound Sepharose CL-4B ( 2 mg / m) were used. Each column is connected in series, prewashed with 10 column volumes of lysis buffer, 10 column volumes of pH 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4 ° C) and then 10 column volumes of lysis Equilibrated with buffer. One human spleen detergent lysate was 0.5-1.0 ml /
Loaded at a flow rate of 1 minute. Two precolumns were used to remove non-specifically bound material from the lysate before passing through the RR1 / 1-Sepharose column.

After loading, RP1 / 1-Sepharose column and bound ICA
M-1 was dissolved in (1) lysis buffer, (2) 20 mM Tris pH 8.0
/0.14M NaCl / 0.1% Triton X-100, (3) 20 mM glycine pH 10.0 / 0.1% Triton X-100, and (4) 50 mM triethylamine pH 11.0 / 0.1% Triton X-100, each having a minimum of 5 column volumes. And washed at a flow rate of 1 m / min. All wash buffers contained 1 mM PMSF and 0.2 μm / m aprotinin. After washing, the residual bound ICAM-1 was added to 5 column volumes of elution buffer (50 mM triethylamine / 0.1%
Elution with Triton X-100 / 4 ° C at pH 12.5) at a flow rate of 1 m / 3 min. The eluted ICAM-1 was collected in a 1 m fraction and immediately neutralized by adding 0.1 m of 1 M Tris, pH 6.7. I
Fractions containing CAM-1 were aliquoted in 10 μl aliquots by SDS-polyacrylamide electrophoresis [Springer et al., JE.
xp.Med. 160 : 1901 (1984)], followed by silver staining [Morri
ssey, JH, Anal . Biochem . 117 : 307 (1981)]. Under these conditions, the bulk of ICAM-1 eluted to approximately one column volume and was more than 90% pure as judged by silver staining electrophoresis (a small amount of IgG leaked from the affinity matrix was a major impurity. T). Fractions containing ICAM-1 were pooled and centricon-30 microconcentrator (Amicon, Denvers, M.M.
It was concentrated approximately 20-fold using A). Purified ICAM-1 was quantified by the Lowry protein assay of an ethanol precipitated aliquot of the pool. About 500 μg of pure ICAM-1
Could be prepared from 200 g of human spleen.

Approximately 200 μg of purified ICAM-1 was subjected to a second stage of purification by preparative SDS-polyacrylamide gel electrophoresis. I c
The band representing AM-1 was visualized by soaking the gel in 1M KCl. The gel region containing ICAM-1 was examined and described in "Hunkapiller et al., Meth. Enzymol. 91: 227-236.
(1983) ". The purified protein was> 98% pure as judged by SDS-PAGE and silver staining.

Affinity Purification of ICAM-1 for Sensory Testing ICAM-1 for sensory testing was purified from detergent lysate from JY cells as described above, but with a small scale (1 m RR).
1/1 Sepharose column) with the following modifications.
All solutions contained 50 μM sodium vanadate. After the column was washed with pH 11.0 buffer containing 0.1% Triton X-100, the column was replaced with 0.1% Triton X-100 for 1 hour.
Washed again with 5 column volumes of the same buffer containing% n-octyl-vader-D-glucopyranoside (octylglucoside). Octyl glucoside detergent is ICAM-
Triton X-100 bound to 1 is removed, and Triton X-1 is removed.
Unlike 00, it can then be removed by dialysis. Then ICAM
-1 was eluted with pH 12.5 buffer containing 1% octyl glucoside instead of 0.1% Triton X-100, analyzed and concentrated as described above.

Example 15 Properties of Purified ICAM-1 ICAM-1 purified from human spleen permeates as a broad band of Mr 72,000-91,000 in SDS-polyacrylamide gel. ICAM-1 purified from JY cells was also from Mr 76,500 to 97,0
Penetrates as a wide band of 00. These Mrs are in the reported range of ICAM-1 immunoprecipitated from various cell sources: Mr from JY cells = 90,000, 114,000 on osteoporotic monocytic cell line U937, and 97,000 on fibroblasts [ Dustin, JI
mmunol . 137 : 245 (1986)]. This broad range of Mr contributes to an extensive but variable degree of glycosylation. The non-glycosylated precursor has a Mr of 55,000 (Dustin
etc). Protein purified from JY cells or human spleen
Its ability to recombine to its original affinity column also
It retains its antigenic activity as evidenced by immunoprecipitation with RR1 / 1 Sepharose and SDS-polyacrylamide electrophoresis.

To prepare a peptide fragment of ICAM-1, about 200 μg was reduced with 2 mM dithiothreitol / 2% SDS and then alkylated with 5 mM iodoacetic acid. The protein was precipitated with _{ethanol, 0.1M NH 4C03 /0.1mM CaCl 2/} 0.
Re-dissolve in 1% zwitterionic agent (Zwittergent) 3-14 (Calbiochem), 37 ° C with 1% w / w trypsin, 4
The digestion was followed by an additional digestion with 1% trypsin at 37 ° C. for 12 hours. Trypsin heptide was purified by reverse phase HPLC using a 0.4 × 15 cm C4 column (Vydac).
The peptide was eluted with a linear gradient from 0% to 60% acetonitrile in 0.1% trifluoroacetic acid. The selected peptides were subjected to sequence analysis using a gas phase microsequenator (Applied Biosystems). Table 5 shows the sequence information obtained from this test.

Example 16 Cloning of ICAM-1 Gene The gene of ICAM-1 can be cloned using any of a variety of procedures. For example, an oligonucleotide sequence corresponding to the ICAM-1 gene can be identified using amino acid sequence information obtained from sequencing of a trypsin fragment of ICAM-1 (Table 5). In addition, the ICAM-1 gene is
Cloning can also be performed by detecting a clone producing ICAM-1 using the ICAM-1 antibody.

ICAM- by using oligonucleotide probe
Cloning of one gene Gene code [Watson, JD, Molecular Biology of t
he Gene, 3rd edition, WA Benjamin, Merono Park,
CA (1977)] can be used to identify one or more different oligonucleotides, each of which can encode an ICAM-1 tryptic peptide. The possibility that a particular oligonucleotide actually constitutes the actual ICAM-1 coding sequence takes into account abnormal base pairing and the frequency (specifically encoding a particular amino acid) of a particular codon using a real eukaryotic cell It can be evaluated by Such “codon usage rules” are described in “Lathe, R. et al., J. Melec. Biol. 183: 1-12 (19
85). Using Lathe's "codon usage rule", theoretically the "most likely" ICAM-1
A single oligonucleotide or set of oligonucleotides is identified that contains a nucleotide sequence capable of encoding a trypsin peptide sequence (ie, a nucleotide sequence with minimal waste).

Oligonucleotides containing a theoretical "most likely" sequence that can encode an ICAM-1 fragment are complementary oligonucleotides or hybrid oligonucleotides that can hybridize to the "most likely" sequence or set of sequences using the set of oligonucleotides. Identify the sequence of the set of oligonucleotides. Oligonucleotides containing such complementary sequences can be used as probes to identify and isolate the ICAM-1 gene [Maniatis, T. et al., Moleculer Cloning A Lab.
oratory Manual, Cold Spring Harbor Press, Cold Spring, NY (1982)].

As described in section C of the above document, IC
From eukaryotic DNA preparations potentially containing the AM-1 gene, I
It is possible to clone the CAM-1 gene. I
To identify and clone the gene encoding the CAM-1 protein, a DNA library is screened for its ability to hybridize to the above oligonucleotide probes. ICAM in normal diploid cells
Genomics is preferred because it appears that only two copies of the -1 gene are present, and because the ICAM-1 gene may have large non-transcribed intervening sequences (introns) where cloning is not desired. The isolation of the ICAM-1 coding sequence from a cDNA library prepared from mRNA of ICAM-1-producing cells rather than DNA. Suitable DNA or cDNA preparations are enzymatically cleaved, randomly sheared, and ligated into a recombinant vector.
The ability of these recombinant vectors to hybridize with the above-described oligonucleotide probes is then measured. Hybridization procedures are described, for example, in “Maniatis, T., Molecu
lar Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) "or" Haymes, BT, etc., Nucleic A
cid Hybridization a Practical Approach, IRL Press, Oxford, UK (1985) ". Analyzing vectors known to be capable of such hybridization, the ICAM-
Analyze the degree and nature of one sequence. According to purely hypothetical considerations, a gene that encodes the ICAM-1 molecule could be unambiguously identified using an oligonucleotide having only 18 nucleotides (by hybridization screening).

That is, in summary, the actual identification of an ICAM-1 peptide sequence allows the identification of a theoretical "most likely" DNA sequence or set of such sequences that can encode such a peptide. By constructing an oligonucleotide complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the "most likely" set of oligonucleotides, ICAM-
DN that can function as a probe to identify and isolate one gene
An A molecule (or set of DNA molecules) is obtained.

Using the ICAM-1 peptide sequence in Table 5, AA and J
The "most likely" sequence of the oligonucleotides that could encode the peptide was determined (Tables 6 and 7, respectively). Oligonucleotides complementary to these sequences were synthesized and purified, and used as probes to isolate the ICAM-1 gene sequence. Select the appropriate size cDNA library by PMA
Prepared from poly (A) ⁺ RNA from induced HL-60 cells and from ps-stimulated umbilical vein endothelial cells. The size-selection cDNA library is described in "Gubler, U. et al. [ Gene 25 : 263-269 (198
3)] and Corbi, A. et al. [ EMBO J. 6 : 4023-4028 (198
7)], using poly (A) ⁺ RNA from PMA-induced HL-60 cells. These documents are incorporated herein by reference.

A size-selected cDNA library was prepared using poly (A) ⁺ from navel vein endothelial cells stimulated with 5 μg / m PS for 4 hours. RNA was extracted by homogenizing the cells in 4M guanidinium isocyanate and subjecting the supernatant to ultracentrifugation with a CsCl gradient (Chirgwin, JM et al., Biochem. 1
8 : 5294-5299 (1979)]. Poly (A) ⁺ RNA was obtained from a mixture of total RNA spaces by oligo (dT) -cellulose chromatography (Type 3, Collaborative Research)
(Aviv, H. et al., Proc. Natl. Acad. Sci.
(USA) 69 : 1408-1412 (1972)].

The first strand cDNA was synthesized using 8 μg of poly (A) ⁺ RNA, avian osteoblastosis virus reverse transcriptase (Life Sciences) and oligo (dT) primers.
DNA-RNA hybrid is digested with Lanase H (BRL),
The second strand was synthesized using DNA polymerase I (New England Biolabs). Ec the product
oR I Linker (New England Biolabs)
At the EcoR I linker (New England)
Biolabs, Inc.)
Digested with I and size selected on low melting agarose gel.
A cDNA larger than 500 bp was ligated to λgt10 which had been digested with EcoRI and dephosphorylated beforehand (Stratagene). The ligation product was then packaged (Stracta Gene Gold).

Next, umbilical vein endothelial cells and the HL-60 cDNA library were spread on a 20,000 PF / 150 mm plate. Recombinant DN
Transfer A to a nitrocellulose filter during replication and add 0.5M
Denatured in NaOH / 1.5M NaCl, 1M Tris, pH7.5 / 1.5M NaC
and baked at 80 ° C. for 2 hours [Benton, W .;
D. et al., Science 196: 180-182 (1977)]. Pre-hybridize the filters, 5x Denhardt solution, 50 mM NaPO ₄
And in 5 × SSC containing 1 μg / m salmon sperm DNA. Prehybridization was performed at 45 ° C. for 1 hour.

Hybridization is 32 bp ('5-TTGGGCTGGTCACAGGAGGT
GGAGCAGGTGAC) or 47 bp (5'-GAGGTGTTCTCAAACAGC
TCCAGGCCCTGGGGCCGCAGGTCCAGCTC) antisense oligonucleotides were used in the manner described above, respectively, on ICAM-1 tryptic peptides J and AA (Tables 6 and 7) [Lathe, RJ Melec. Biol . 183 : 1-12 ( 198
Five)〕. Oligonucleotides were end-labeled using the recommended conditions r- ⁽³² P) T ⁴ polynucleotide kinase and manufacturers with ATP (New England Biolabs). Following overnight hybridization, the filters were washed twice at 45 ° C. for 30 minutes with 2 × SSC / 0.1% SDS. Phages were isolated from plaques showing hybridization and purified by serial recoating and rescreening.

Cloning of ICAM-1 Gene Using Anti-ICAM-1 Antibody The ICAM-1 gene can alternatively be cloned using an anti-ICAM-1 antibody. DNA or more preferably c
DNA is extracted and purified from cells capable of expressing ICAM-1.
Fragment the purified cDNA (by shearing, endonuclease digestion, etc.) to prepare a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are cloned into an expression vector to prepare a gene library of expression vectors, each member containing a specific cloned DNA or cDNA fragment.

Example 17 Analysis of cDNA Clones Phage DNA from positive clones was digested with EcoR I,
The cDNA from one clone was tested by Southern analysis using a probe. The cross-hybridized maximum size cDNA insert was subcloned into the EcoRI site of plasmid vector pGEM4Z (Promega). HL-60 subclone containing cDNA in both orientations was digested with endonuclease III [ Henikoff, S., Gene 28 : 351-359 (1984)].
According to the manufacturer's recommendations (Erasur Base, Blomega). The progressively deleted cDNA was then cloned and subjected to dideoxynucleotide chain-end sequencing according to the manufacturer's recommendations (Sequenase, US Biochemical) [Sanger, F. et al., Proc . N
atl . Acad . Sci (USA) 74 : 5463-5467 (1977)]. HL−
The 60 cDNA 5 'and coding region were completely sequenced on both strands and the 3' region was approximately 70% on both strands.
Sequenced. A representative endothelial cDNA was sequenced over most of its length by shotgun cloning of a 4 bp recognition restriction enzyme fragment.

The cDNA sequences of one HL-60 and one endothelial cell cDNA were established (FIG. 8). The 3023 bp sequence has a short 5 'untranslated region and a common polyadenylation signal at position 2966.
Includes 3kb 3 'untranslated region. The longest open reading frame begins with the first ATG at position 58 and ends with the TGA end triplet at position 1653. An ICAM-1 cDNA clone was isolated whose identity between the just translated amino acid sequence and the sequence determined from eight different tryptic peptides of a total of 91 amino acids (underlined in FIG. 8) I realized that it was. The amino acid sequence of ICAM-1 trypsin peptide
It is shown in the table.

Hydrophobicity analysis (Kyte, J. et al., J. Melec . Biol . 157 : 105-132.
(1982)] suggests the presence of a 27 residue signal sequence. The role of +1 glutamine is consistent with the inability of the present invention to obtain N-terminal sequences on three ICAM-1 protein preparations; Bring. The simple sequence of 1-453 is primarily hydrophilic, followed by a putative transmembrane domain of 24 residues hydrophobic. Transmebranding is immediately followed by several charged residues contained within the 27 residue putative cytoplasmic domain.

The suggested size of the mature polypeptide chain is 55,219 daltons and the observed size of deglycosylated ICAM-1 is 55,055.
Good match with 00 (Dustin, ML, et al. , J. Immunol.
137 : 245-254 (1986)]. Eight N-linked glycosylation sites are predicted. The absence of asparagine in the two tryptic peptide sequences at these sites confirms their glycosylation and their extracellular orientation. Assuming 2,500 daltons per high mannose N-linked carbohydrate, a size of 75,000 daltons was predicted for the ICAM-1 precursor, and the observed size of 73,000 daltons (Dustion, ML et al., J. Immunol. 137: 245-254). (19
86)]. After conversion of high mannose to complex carbohydrate, the mature ICAM-1 glycoprotein is 76-114 kd, depending on the cell type (Dustin, ML et al., J. Am.
Immunol. 137: 245-254 (1986)]. That is, ICAM-1 is highly glycosylated but is a typical complete membrane protein.

Example 18 ICAM-1 is an integrin-binding member of the immunoglobulin supergene family: ICAM-1 internal repeats were cloned into Microgeni
e) Protein sequence program [Queen, C. et al., Nucl.
es. 12: 581-599 (1984)]. The sequence of ICAM-1 for IgM, N-CAM and MAG was determined using the Microdini and ALIGN programs [Daypof
f, MO, et al., Meth. Enzmol. 91: 524-545 (1983)]. National Biomedical Research
Foundation (National Biomedical Research Fou
ndation) was searched for protein sequence similarity using Williams and Pearson's FASTP program [Lipman, DJ
Et al., Science 227 : 1354-1439 (1985)].

Since ICAM-1 is a ligand for integrins, it was not expected to be a member of the immunoglobulin supergene family. However, examination of the ICAM-1 sequence has shown that the sequence meets all the proposed criteria for family relationships in immunoglobulin supergenes. These criteria are described below.

The entire extracellular domain of ICAM-1 is constructed from the five homologous immunoglobulin-like domains shown in FIG. 9A. Domains 1-4 are 88, 97, 98 and 102 residues long, respectively, and thus have a typical Ig domain size; domain 5 is truncated at 68 residues. A search of the NBRF database using the FASTP program reveals significant homology with members of the immunoglobulin supergene family, including the IgM and IgG C domains, the T cell receptor α subunit variable domain, and the alpha1beta glycoprotein. (FIGS. 9B-D).

Using the above information, the amino acid sequence of ICAM-1 was compared with the amino acid sequence of another member of the immunoglobulin supergene family.

The three types of Ig supergene domains, V, C1 and C2, are differentiated. V and C domains are linked together by an internal domain disulfide bond
-Constructed from sheets; V domains have 9 antiparallel β-strands and 7 C domains. The constant domains were divided into C1 and C2-sets based on the characteristic residues shown in FIG. 9A. The C1-set contains the proteins involved during antigen recognition. C2--set contains several F _C
It contains receptors and proteins contained in cell adhesions including CD2, LFA-3, MAG and NCAM. ICAM−
One domain replaces ICAM-1 in this set
It was found to have the strongest homology with the C2-set domain; this is more similar to the retained residues in C2 than to the C1 domain as shown for β-strand BF in FIG. It is reflected in what you do. In addition, the ICAM-1 domain is more likely to be the β strands A and G of the C2 domain
And better alignment, allowing better alignment across the entire C2 domain intensity. The sequence with the C2 domain from NCAM, MAG and the alpha1-beta glycoprotein
Shown in FIGS. 9B and 9C; identities ranged from 28 to 33%. T cell receptor Vα27% identity and
The sequence with IgM C domain 3 34% identity is also shown (Figures 9B, 9D).

One of the most important features of the immunoglobulin domain is β
Disulfide-bonded cysteines bridging the B and Fβ strands that stabilize the sheet sandwich; ICAM
At -1 the cysteine is retained in all cases, but at strand f of domain 4 it faces the sandwich and some other V- and C-2
Leucine has been found to stabilize contacts as proposed in the set domain. Sistine (43, 5
The distance between (0, 52 and 37 residues) is as described for the C2 set.

To test for the presence of interchain disulfide bonds in ICAM-1, endothelial cells ICAM-1 were subjected to SDS-PAGE under reducing and non-reducing conditions. Endothelial cells ICAM-1
Y or hairy spleen cells were used because they show less glycosylation heterogeneity than ICAM-1 and show greater sensitivity to shifts in Mr. Therefore, ICAM-1 was converted to LPS for 16 hours.
(5 μg / m) Stimulated navel vein endothelial cell culture was purified by immunoaffinity chromatography as described above. Sample buffer containing acetone precipitated ICAM-1 containing 0.25% 2-mercaptoethanol or 25 mM iodoacetamide [ Lacemmli, UK, Nature 227 : 680-685
(1970)] and brought to 100 ° C. for 5 minutes.
The sample was subjected to SDS-PAGE4760 and silver staining 4613.
Endothelial cell ICAM-1 showed an apparent Mr of 100 kd under reducing conditions and 96 kd under non-reducing conditions, strongly suggesting the presence of interchain disulfide in native ICAM-1.

The use of primary sequences to predict secondary structure [Chon, PY et al., Biochem . 13 : 211-245 (1974)]
In each of the ICAM-1 domains, labeled ag at the top of FIG. 9A and corresponding to each position of strand AH in the immunoglobulin (bottom of FIG. 9A), exactly meeting the expectations for the immunoglobulin domain. It showed seven expected β-strands. Although domain 5 lacks the A and C strands, but these form the ends of the sheets, each sheet is still probably formed by strand D and has been proposed in several other C2 domains As such, it represents strand C; the characteristic disulfide bond between B and F strands will not be affected. That is, the domain size, sequence homology, the retention cysteine that forms a putative interdomain disulfide bond, the presence of disulfide bonds, and the criteria for predicted β-sheet structure are all ICAM-
Satisfies the intrinsic of 1.

ICAM-1 was found to be most strongly homologous to the C2 set of NCAM and MAG glycoproteins. This is what NCAM
Of particular interest is that MAG both mediates cell-cell adhesion. NCAM is important in neuron-neuron and neuro-muscle interactions [Cunningham, BA et al., Science
236 : 799-806 (1987)], and MAG is myelinated (myeli
neutron-oligodentrocyte and oligodentrocyte-oligodentrocyte interactions [Poltorak, M. et al., J. Cell . Biol .
5 : 1893-1899 (1987)]. Cell surface expression of NCAM and MAG is developmentally regulated during nervous system formation and myelimination, respectively, similar to the regulated induction of ICAM-1 in inflammation [Springer, TA et al., Ann . Rev. Immunol. .5 : 223-25
2 (1987)]. ICAM-1, NCAM [Cunningham, BA, etc., Sc
ience236 : 799-806 (1987)], and MAG [Salzer, J.
L. et al., J. Cell. Biol . 104 : 957-965 (1987)] are similar and homologous in overall structure. This is because each is a complete membrane glycoprotein composed of five C2 domains that form the N-terminal extracellular region. However, NC
In AM, some additional non-Ig-like sequences are present between the last C2 domain and the transmebranding domain. ICAM-1
Is sequenced with 21% identity to MAG over its entire length, including the transmembrane and cytoplasmic domains; the same% identity when comparing the five domains of ICAM-1 and NCAM-1 Found. A schematic comparison of the secondary structure between ICAM-1 and MAG is shown in FIG. Domain-to-domain comparisons indicate the level of homology between ICAM-1 and the domain within the NCAM molecule (× ± sd21 ± 2.8% and 18.6 ± 3.8, respectively).
%) Indicates the same level of homology when comparing the ICAM-1 domain to the NCAM and MAG domains (20.4 ± 3.7 and 21.9 ± 2.7, respectively). NCAM
[Cunningham, BA et al., Science 236 : 799-806 (198
7)]; Barthels, D. et al., EMBO J. 6 : 907-914 (1987)]
And MAG [Lai, C. et al., Proc. Natl. Acad. Sci. (USA) 84 :
4377-4431 (1987)], although there is evidence of additional conjugation in the C-terminal region,
Studies of the ICAM-1 protein backbone and precursors in the sequencing of AM-1 clones or of various types [Dustin, ML et al. , J. Immunol. 137: 245-25
4 (1986)].

ICAM-1 functions as a ligand for LFA-1 in lymphocyte interactions with many different cell types. Lymphocytes bind to ICAM-1 contained in the artificial membrane bilayer, which requires LFA-1 on the lymphocytes and directly suggests an interaction between ICAM-1 and LFA-1 (Marlin, SD Etc., Cell 51: 8
13-819 (1987)]. LFA-1 is a leukocyte integrin and has no immunoglobulin-like characteristics. Leukocyte integrins contain one integrin subfamily. The other two subfamilies mediate cell matrix interactions and recognize the sequence REG within their ligands, including fibronectin, vitronectin, collagen and fibrinogen [Hyn
es, RO, Cell 48 : 549-554 (1987)]; Ruoslahti, E. et al.
Science 238 : 491-497 (1987)]. Leukocyte integrins are expressed only on leukocytes, are involved in cell-cell interactions, and only a few known ligands are ICAM-1 and iC3b (ie, a fragment of complement component C3 that does not exhibit immunoglobulin-like characteristics) -1 [Kishimoto,
TK, Leukocyte Typing III , McMi Chael, M. Ed., Springer Belllag New York (1987); Springe
r, TA, et al., Ann . Rev. Immunol. 5 : 223-252 (1987); Ander
Son , DC et al., Ann . Rev. Med. 38 : 175-194 (1987)]. By sequence analysis, potential peptides within the ICAM-1 sequence recognized by LFA-1 are shown in Table 9.

ICAM-1 is the first example of a member of the immunoglobulin supergene family that binds to integrins. Although both of these groups play important roles in cell adhesion, the interaction between the two was not previously expected. In contrast, interactions within immunoglobulin supergenes are quite common. It is quite possible that further examples of interactions between integrins and immunoglobulins will be found. LFA-1 recognizes a ligand different from ICAM-1 [Springer, TA et al. , Ann. Rev. Immunol. 5 : 223-2 .
52 (1987)], leukocyte integrin Mac-1 is a neutrophil-
Recognizes a different ligand from C3bi on neutrophil adhesion [Anders
on, DC, et al. , Ann. Rev. Med. 38 : 175-195 (1987)]. Furthermore, the purified MAG-containing vesicles (vesicles) bind to the MAG neurites, so that MAG must be capable of heterophilic interaction with heterologous receptors [Poltorak, M. et al., J. Am. Cell. Biol. 105 : 1893-1899 (1
987)].

NC in nerve-nerve and nerve-muscle cell interactions
It has been suggested that the role of AM is based on homophilic NCAM-NCAM interactions [Cunningham, BA et al., Science 236 :
799-806 (1987)]. The critical role of MAG in the interaction between the nearby rotating loops of Chevane cells that take up axons during myelin sheath formation may be based on interactions with heterologous receptors or homophilic MAG-MAG interactions. NCAM homology and frequent occurrence of domain-domain interactions within immunoglobulin supergenes may indicate that ICAM-1 may be involved in homophilic and ICAM-1-LFA-1 heterophilic interactions Cause sex. However, binding of B lymphoblastoid cells co-expressing LFA-1 and ICAM-1 at similar densities to ICAM-1 in artificial or cell monolayers is
Pretreatment of lymphoblasts with LFA-1 MAb can be completely suppressed, but adhesion is not affected by B-lymphoblast pretreatment with ICAM-1 MAb. Pretreatment of the monolayer with ICAM-1 Mab completely destroyed the bond [Du
Stin, ML et al. , J. Immunol. 137: 245-254 (1986); Marli
n, SD, Cell , 51 : 813-819 (1987)]. These findings indicate that if any ICAM-1 homophilic interaction occurs,
It indicates that the effect must be much weaker than the heterophilic interaction with LFA-1.

The possibility that leukocyte integrins recognize ligands in fundamentally different ways is important in ligand binding and RG
D is consistent with the presence of a 180 residue sequence in their α subunit that is not present in cognitive integrins (Corbi, A. et al.
EMBO J. 6: 4023-4028 (1987)]. Mac-1 is iC3b50
Although it has been proposed to recognize the RGD sequence present in 86, there is no RGD sequence in ICAM-1 (No. 8).
Figure). This is consistent with the inability of the fibronectin peptide GRGDSP and control peptide GRGESP to suppress ICAM-1-LFA-1 adhesion [Marlin, SD et al., Cell . Five
1 : 813-819 (1987)]. However, PRGGS and RGE
Related sequences such as KE are found in ICAM-1 in β-strands a and b of domain 2 and c and d of domain 2, respectively.
Exists in the region foreseen in the loop between (FIG. 9) and is therefore acceptable for recognition. Of interest is that the homologous MAG molecule contains an RGD sequence between domains 1 and 2 [Poltorak, M. et al., J. Cell . Biol .
5 : 1893-1899 (1987); Salzer, JL et al. , J. Cell. Biol.
104 : 957-965 (1987)].

Example 19 Southern and Northern Blots Southern blots were extracted from 5 cell lines from 3 cell lines.
g2 gene DNA: BL2, Burkitt's lymphoma cell line (provided by Dr. Gilbert Lenoir); JY and Er
-EBV transformed B-lymphoma-like cell line of LCL.

Each DNA was digested with 5 × manufacturer recommended amounts of BamH1 and EcoRI endonuclease (New England Biolabs). Following electrophoresis on a 0.8% agarose gel, the DNA was transferred to a nylon membrane (Zeta Probe, Bio-Rad). ICAM from HL-60 labeled with pre-hybridized filter and α- ( ³² P) d XTP's
Hybridization was performed by random priming according to standard procedures using cDNA. Northern blot 20μ
g of total DNA or 6 μg of poly (A) ⁺ RNA. The DNA was denatured, electrophoresed on a 1% agarose-formaldehyde gel, and electrophoresed to a zeta probe. Each filter was pre-hybridized and hybridized as above using a ³² P-labeled oligonucleotide probe (described above) HL-60 cDNA probe (Stau).
Embo J. 6 : 3695-3701 (1987)].

Southern blots using a 3 kb cDNA probe and genomic DNA digested with BamHI and EcoR1 show single genes of 20 and 8 kb, respectively, indicating that most of the coding information is within 8 kb. A hybridizable fragment was indicated. There was no evidence of restriction fragment polymorphism in the blots of the three cell lines.

Example 20 ICAM-1 Gene Expression An "expression vector" can express DNA (or cDNA) that is cloned into a vector (based on the presence of appropriate transcriptional and / or translational control sequences), A vector capable of producing a polypeptide or protein. Expression of the cloned sequence occurs when the expression vector is integrated into a suitable host cell. If a prokaryotic expression vector is used, a suitable host cell will be any prokaryotic cell capable of expressing the cloned sequence. Similarly, when using eukaryotic expression vectors, suitable host cells are any eukaryotic cells capable of expressing the cloned sequence. Importantly, to produce a prokaryotic genomic expression vector library, ICAM-1 must be expressed since eukaryotic DNA can contain intervening sequences and such sequences cannot be processed accurately in prokaryotic cells. It is preferred to use cDNA from a potential cell.

Methods for preparing cDNA and producing genomic libraries are disclosed by Maniatis, J. et al. [Molec
ular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)].

A bank of host cells is made using the expression vector gene library described above, each of which contains one member of the library. The expression vector can be incorporated into the host cell by any of a variety of means (ie, transformation, transfection, protoplast fusion, electroporation, etc.). The bank of expression vector containing cells is grown clonally and each member is assayed individually (using an immunoassay) to determine whether they produce a protein that can bind to the anti-ICAM-1 antibody.

Expression vectors for cells producing proteins capable of binding to the anti-ICAM-1 antibody were further analyzed and these vectors were
Whether to express (or contain) the ICAM-1 gene, IC
Express (or contain) only a fragment of the AM-1 gene
To determine whether the product expresses (or contains) a gene that is not ICAM-1, even if the product is immunologically related to ICAM-1. Although such analysis may be performed in any convenient way, it is preferable to determine the nucleotide sequence of the DNA or cDNA cloned into the expression vector. Such nucleotide sequences are examined to determine if they can encode a polypeptide having the same amino acid sequence as the tryptic digested fragment of ICAM-1 (Table 5).

An expression vector comprising a DNA or cDNA molecule encoding the ICAM-1 gene is thus capable of (i) expressing a protein capable of binding to an anti-ICAM-1 antibody; and (ii)
It can be recognized by the presence of a nucleotide sequence that can encode each of the trypsin fragments of ICAM-1. The cloning DNA molecule of such an expression vector can be removed from the expression vector and isolated in pure form.

Example 21 Functional activity of purified ICAM-1 In cells, ICAM-1 normally functions as a surface protein associated with the cell membrane. Therefore, the function of purified ICAM-1 was tested after reconstitution of the molecule by dissolving the protein in an artificial lipid membrane (liposomes or vesicles) in detergent-solubilized lipids and removing the detergent by dialysis. . ICAM-1 purified from JY cells and eluted as described above in detergent octyl glycoside is reconstituted in vesicles, and vesicles containing ICAM-1 are fused to glass cover slips or plastic culture wells and bound to protein. Cells to be detected.

Preparation of flat membrane and plastic-bound vesicles Vesicles were prepared by the method of Gay et al. [ J. Immunol.
136 : 2026 (1986)]. That is, egg phosphatidyl chlorin and cholesterol were dissolved in chloroform and 7: 2
At a molar ratio of The lipid mixture was dried to a thin film while rotating under a stream of nitrogen gas and then lyophilized for 1 hour to remove any traces of chloroform. The lipid membrane was washed with 1% octyl glycoside / 0.14 M NaCl / 20 mM Tris (pH
In 7.2), phosphatidylchlorin was dissolved to a final concentration of 0.1 mM. About 10 μg of purified ICAM-1 or human glycophorin (Sigma Chemical Co., St. Louis, Mo.) as a control membrane glycoprotein was added to each m of the dissolved lipid.
The protein-lipid-detergent solution was added to 200 volumes of 20 mM Tris / 0.
Dialysis was performed at 4 ° C. against three changes of 14M NaCl, pH 7.2 and one change of HBSS.

A flat film was prepared by the method of Brian et al . [ Proc.
cad.Sci.81 : 6159 (1984)]. Glass coverslips (diameter 11 mm) are diluted 1: 1 in a 1: 6 dilution of 17x detergent (Linbro).
Boiled for 5 minutes, washed overnight in distilled water, immersed in 70% ethanol, and air-dried. An 80μ droplet of a vesicle suspension containing either ICAM-1 or Clicophorin was placed in the bottom of a well of a 24-well Culster plate, and the glass coverslip prepared above was gently floated on top.
After a 20-30 minute incubation at room temperature, each well is
Filled with HBSS and flipped the coverslip so that the flat surface was facing up. The wells were then washed extensively with HBSS to remove unbound vesicles. The flat film surface was not exposed to air at all.

During the experiment with the flat film fused to the glass surface, the IC
Vesicles containing AM-1 bound directly to the plastic surface of the multiwell tissue culture plate and were found to retain functional activity as evident by specific cell binding. Such vesicles are hereinafter referred to as “Plastic-Bound Vesicles (PBV), because they do not measure the properties of lipid vesicles bound to plastic. Prepared by adding to the bottom of wells in a 96-well tissue culture tray (Falcon) followed by incubation and washing as described for flat membranes.

Cell Adhesion Assay Both cell adhesion assays using flat membranes or plastic-bound vesicles were performed in essentially the same way, but with PB
The cell number and volume of the V assay was reduced to 1/5 that used in the flat membrane assay.

Normal controls and leukocyte adhesion deficiency (LAD) patients who cannot express LFA-1 [Anderson, DC et al., J. Infect.
is . 152 : 668 (1985)] at 1 μg / m
Prepared by culturing in peripheral blood mononuclear cells RPMI-1640 + 20% FCS containing 5 × 10 ⁵ cells / m for 3 days in Concanavalin-A (Con-A). The cells were then incubated with RPMI for 2 hours.
Times; once with 5 mM methyl-alpha-b-manophilanoside to remove residual lectin from the cell surface. Cells
The cells were grown in RPMI / 20% FCS containing 1 ng / m recombinant IL-2 and used between 10 and 22 days after the start of culture.

To detect cells that bind to flat membranes or PBV, use Con
A blasts, T-lymphoma SKW-3, EBV transformed B- lymphoma-like cell line JY (LFA-1 positive) and LFA-1-deficient lymphoma-like cell line (BBN) [from a patient 1, Springer, TA et al., J .
Exper.Med.160 : 1901-1918 (1986)] at 1m RPMI-16
1 × 10 ⁷ cells in 40/10% FCS were treated with 100 μCi Na ⁵¹ CrO ₄
C. for 1 hour, then radioactively labeled by washing four times with RPMI-1640 to remove unbound label. In the monoclonal antibody blocking test, cells or plastic-bound vesicles were RPMI-1640
Pretreatment with 20 μg / m of purified antibody in 10% FCS at 4 ° C. for 30 minutes, followed by four washes to remove unbound antibody. In experiments of the effect of divalent ions on cell binding,
The cells were washed once with HBSS without Ca ²⁺ and Mg 2 ⁺⁺ 10% dialyzed FCS, and CaCl and MgCl were added to the determined concentrations. In all experiments, cells and flat membranes or PBV were pre-equilibrated in the appropriate assay buffer at the appropriate temperature (4 ° C, 22 ° C or 37 ° C).

To determine cells that bind to purified ICAM-1, ⁵¹ Cr
Labeled cells (5 × 10 ⁵ EBV transformants in flat membrane assay; P
1 × 10 ⁵ EBV transformants or SKW-3 cells in a BV assay, 2 × 10 ⁵ Con-A blasts) on a flat membrane or PBV at 25 × g
And incubated at 4 ° C, 22 ° C or 37 ° C for 1 hour. After incubation, unbound cells were removed by eight cycles of filling and aspiration with pre-equilibrated buffer at the appropriate temperature. Bound cells were quantified by solubilizing well contents with 0.1N NaOH / 1% Triton X-100 and counting in a gamma counter. The% cell binding was determined by dividing the cpm from the bound cells by the cpm of the input cells. In a flat membrane assay, the input cpm was collected relative to the ratio of the surface area of the coverslip to the surface area of the culture well.

In these assays, EBV-transformed B-lymphoma cells, SKW-3T-lymphoma cells, and Con-AT lymphoblasts specifically bound to ICAM-1 in the sclera (FIGS. 11 and 12). . Binding was specific because the cells bound very poorly to control flat membranes or vesicles containing equivalent amounts of other human cell surface glycoprotein glycophorins. Furthermore, LFA-1 positive E
BV transformants and Con A blasts also bound, but their LFA-1 negative equivalent did not bind to any significant degree, indicating that binding was dependent on the presence of LFA-1 on the cells. Was.

Both the specificity of cell binding and the dependence on cell LFA-1 were confirmed in a monoclonal antibody blocking test (FIG. 13). JY cell binding was observed when ICAM-1-containing PBV was pretreated with anti-ICAM-1 monoclonal antibody RR1 / 1.
%. Pretreatment of cells with the same antibody had little effect. Conversely, anti-LFA-monoclonal antibody R
S1 / 18 inhibited binding by 96% but only slightly when cells were pretreated but not PBV. The control antibody TS2 / 9 (different lymphocyte surface antigen) reactive with LFA-3 had no significant inhibitory effect when pretreated with either cells or PBV. This experiment is free of some amount of impurities in the artificial membrane
It shows that ICAM-1 itself mediates the observed cell adhesion and that adhesion is dependent on LFA-1 on binding cells.

Cell binding to ICAM-1 in artificial membranes also demonstrated two other properties of the LFA-1-dependent adhesion system: temperature dependence and the need for divalent cations. As shown in FIG. 14, Con-A blasts bound ICAM-1 in PBV most effectively at 37 ° C., partially at 22 ° C. and very poorly at 4 ° C. As shown in FIG. 15, binding is entirely dependent on the presence of divalent cations. At physiological concentrations, Mg ²⁺ alone showed the highest cell binding, whereas Ca ²⁺ alone showed very low levels of binding. However, ^1/1 of the normal concentration in combination with Ca ²⁺
A Mg ²⁺ of 10 had a synergistic effect and showed the best binding.

In summary, the specificity of cell binding to purified ICAM-1 contained in artificial membranes, specific inhibition by monoclonal antibodies, and the need for temperature and divalent cations
-1 indicates that it is a specific ligand of the LFA-1-dependent adhesion system.

Example 22 ICAM-1 in allergic and toxic patch test reactions
And HLA-DR expression Five normal human skin biopsies were analyzed for their ICAM-1 and HLA-D
Performed for R expression. Endothelial cells in certain blood vessels are usually ICA
Although M-1 was expressed, ICAM-1 was not expressed in keratinocytes from normal skin. No staining of HLA-DR on any keratinocytes from normal skin biopsies was observed. The expression kinetics of ICAM-1 and class II antigens were examined in cells from biopsies of allergic and toxic skin disorders. Half of the six subjects examined were found to have ICAM-1 expressing keratinocytes 4 hours after hapten application (Table 10). Hapten exposure time increases the proportion of people expressing ICAM-1 on keratinocytes and by 48 hours more ICAM / keratinocytes
The staining intensity showing -1 expression also increased. In fact, at this point, a portion of the keratinocytes of all biopsies stained positive for ICAM-1. 72 hours (24 hours after hapten removal)
Thus, seven of eight subjects expressed ICAM-1 in keratinocytes, and one subject had reduced ICAM-1 expression between 48 and 72 hours.

^a sample keratinocytes at least subgroups were considered positive when stained.

^b All patches were removed at this point.

Histologically, the stained image of ICAM-1 on keratinocytes from a biopsy taken 4 hours after the application of the hapten was usually a small colony. After 48 hours, ICAM-1 was expressed on most surfaces of keratinocytes and there was no difference between the center and periphery of the lesion. The staining intensity decreased when the keratinocytes approached the stratum corneum of the nail. It was found in biopsies taken from the center and around the lesion. Also at this time, the patch test was positive (wet, erythema, vesicles). No differences in ICAM-1 expression were seen when different haptens were applied to susceptible individuals. In addition to keratinocytes, IC
AM-1 was also expressed on some mononuclear cells and endothelial cells at the site of injury.

The expression of HLA-DR on keratinocytes of allergic skin disorders was less frequent than the expression of ICAM-1. Among the subjects studied, HLA-DR should be used by 24 hours after hapten application.
None had any damage due to keratinocytes that stained positively. In fact, only four biopsy samples were HLA
-Only have keratinocytes expressing DR, HLA-D
None of the biopsies had keratinocytes positive for R and not positive for ICAM-1 (Table 10).

In contrast to the allergic patch test lesion, the toxic patch test lesion first induced with oil or sodium lauryl sulfate had keratinocytes with little ICAM-1 on its surface at all time points in the test ( Table 11). Indeed, 48 hours after application of the patch, this is the optimal time point in allergic patch test subjects, but one of the 14 toxic patch test subjects has one of the keratins expressing ICAM-1 during a disorder. Had cells. Also, in contrast to allergic patch test biopsies, HLA-DR was not expressed on keratinocytes in toxic patch test lesions.

These data indicate that ICAM-1 is expressed in immune system inflammation and not in toxic system inflammation, thus:
ICAM-1 expression can be used to differentiate immune and toxic inflammation, such as acute renal disease, in renal transplant patients where it is difficult to determine whether the disease is due to rejection of immunosuppressive therapeutics or urinary toxicity. Renal biopsy and evaluation of enhanced ICAM-1 expression will allow the differentiation between immune system rejection and non-immune system toxic reactions.

^a sample was considered positive if the subgroup of at least keratinocytes were stained.

^b All patches were removed at this point.

Example 23 Expression of ICAM-1 and HLA-DR During Benign Skin Disease Cells from injured skin biopsies from patients with various types of inflammatory skin diseases are expressed for their ICAM-1 and HLA-DR expression investigated. Some of the keratinocytes during biopsy of allergic contact eczema, pemphigus, and lichen planus were ICAM-1
Was expressed. Lichen planus showed the strongest staining with images that were comparable or somewhat stronger than those seen in the 48 hour allergic patch test biopsy (Table 12). Similar to the results of the allergic patch test, the strongest ICAM-1 staining was seen at sites of high mononuclear cell infiltration. Furthermore, eight of the eleven lichen planus biopsies tested were positive for HLA-DR expression on keratinocytes.

The expression of ICAM-1 on keratinocytes from skin biopsies of patients with rash and urticaria was low. Only four of the seven patients tested with these diseases had keratinocytes that expressed ICAM-1 at the site of injury. HLA
-DR expression is only in one patient,
-1.

Portions of endothelial cells and mononuclear cell infiltrates from all of the benign inflammatory skin lesions tested expressed ICAM-1 to varying degrees.

^a sample was considered positive if the subgroup of at least keratinocytes were stained.

Example 24 ICAM-1 expression on keratinocytes of psoriatic skin disease ICAM-1 during skin biopsy from 5 patients with psoriasis
Expression was examined periodically before and during PUVA treatment. Biopsies were obtained from five patients with classic psoriasis recognized by histology. Biopsies were taken before PUVA treatment and continuously during the indicated times. PUVA was administered 3-4 times a week. Biopsies were taken from around the psoriatic plaques of five patients, and in addition to biopsies, from four clinically normal skins of four of these patients.

Each fresh skin biopsy was frozen and stored in liquid nitrogen. 6 micron stock sections were air-dried overnight at room temperature, fixed in acetone for 10 minutes, and immediately stored at -80 ° C until stained or wrapped in aluminum foil and stained.

Staining was made in the following manner. The sections were incubated with the monoclonal antibody and stained by a three-step immunoperoxidase method using diaminobenzine H ₂ O ₂ and a substrate [Stein, H. et al., Adv . Cencer Res . 42 : 67-147, (198
Four)〕. Anti-ICAM-1 and HLA in tonsils and lymph nodes
-Used as a positive control for DR staining. Tissue stained in the absence of primary antibody was a negative control.

Becton Dickin monoclonal antibody against HLA-DR
Purchased from son (Mountain View, CA). The anti-ICAM-1 monoclonal antibody was R6-5-D6. Peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjugated pig anti-rabbit Ig were purchased from DAKAPATTS, Copenhagen, Sweden. Diaminobenzidine-tetrahydrochloride was obtained from Sigma (St. Louis).

The results of the study showed that endothelial cells of some blood vessels expressed ICAM-1 in both diseased and normal skin,
Staining intensity and the number of vessels expressing ICAM-1 were increased in psoriatic skin lesions. Furthermore, the expression pattern of ICAM-1 in keratinocytes of untreated psoriatic skin lesions from 5 patients changed from a small group of cell staining to a large number of keratinocytes. During the course of PUVA treatment, ICAM-1 expression in two patients (patients 2 and 3) showed a significant reduction prior to or at the same time as clinical remission. Patients 1, 4 and 5 were associated with clinical relief or worsening, respectively.
ICAM-1 expression was reduced or increased during PUVA treatment.
ICAM on keratinocytes from normal skin before and after PUVA treatment
-1 expression was absent. This indicates that PUVA does not induce ICAM-1 on keratinocytes from normal skin.

Of note, mononuclear cell infiltration density was correlated with ICAM-1 expression on keratinocytes, which indicates that mononuclear cells during injury during PUVA treatment when ICAM-1 expression also diminished. Reduced cell number and IC on keratinocytes
It is associated with both an increase in the number of mononuclear cells during PUVA treatment when AM-1 expression becomes more pronounced. Endothelial cells and skin mononuclear cells are also ICAM-1 positive. In clinically normal skin, ICAM-1 expression was found in endothelial cells without keratinocyte labeling.

The expression of HLA-DR on keratinocytes was variable. I
There were no non-CAM-1 positive HLA-DR positive biopsies.

In summary, these results indicate that before treatment, ICAM-1
Expression is high on keratinocytes, indicating that it correlates with mononuclear cell infiltration density. During PUVA treatment, a significant decrease in ICAM-1 staining is seen in parallel with clinical improvement. Histologically, skin infiltration had disappeared. If clinical deterioration was seen during treatment, ICAM-1 expression on keratinocytes as well as skin infiltration density was increased. When clinical relief was seen during treatment, there was a concurrent decrease in ICAM-1 staining on keratinocytes as well as a decrease in skin infiltration. That is, the expression of ICAM-1 on keratinocytes corresponded to the density of mononuclear cells infiltrating the skin. These data indicate that the clinical response to PUVA treatment results in an accelerated decrease in ICAM-1 expression on keratinocytes in parallel with a more gradual decline in mononuclear cells. This indicates that ICAM- on keratinocytes
1 shows that expression is responsive to onset and persistence of skin invasion, and that PUVA treatment down-regulates ICAM-1 and alleviates skin invasion and inflammatory response. The data also shows that HLA-DR on keratinocytes during PUVA treatment was upregulated.

ICAM-1 expression on keratinocytes of psoriatic lesions correlates with the clinical severity of the lesion and the size of skin invasion. Thus, ICAM-1 plays a central role in psoriasis, and its suppression and / or its interaction with the CD18 complex on mononuclear cells would be an effective treatment for this lesion. Furthermore, ICAM-1 on keratinocytes
Monitoring expression will be an effective tool for diagnosing, preventing, and assessing the course of treatment for psoriasis.

Example 25 Expression of ICAM-1 and HLA-DR During Malignant Skin Disease Unlike benign skin lesions, the expression of ICAM-1 on keratinocytes from malignant skin injury was variable (Table 14). ). Of the 23 skin T-cell lymphomas tested, ICAM-1-positive keratinocytes were identified in only 14 cases. Keratinocytes from biopsy of mycosis fungoides lesions show that ICAM as their disease progresses to earlier stages
-1 expression tended to digest. However, ICAM
-1 expression was seen on mononuclear cell infiltrates at a varying rate from most of the skin T-cell lymphoma lesions. Of the remaining lymphomas tested, four out of eight had keratinocytes that expressed ICAM-1. Of the 29 patients with malignant skin malignancies tested, 5 had keratinocytes that expressed HLA-DR without expressing ICAM-1 (first
4 Tables).

^a Each sample was considered positive if at least a small group of keratinocytes was stained.

Example 26 Effect of Anti-ICAM-1 Antibody on Proliferation of Human Peripheral Blood Mononuclear Cells Human peripheral blood mononuclear cells are proliferated and induced by the presence and recognition of an antigen or mitogen. Mitogens, some molecules, such as concanavalin A or T cell-binding antibody OKT ₃ causes nonspecific proliferation of peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells are heterogeneous in that they consist of a subpopulation of cells that can recognize specific antigens. When a peripheral blood mononuclear cell capable of recognizing a specific specific antigen encounters that antigen, the proliferation of the subpopulation of mononuclear cells is induced. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens recognized by a subpopulation of peripheral mononuclear cells but are not recognized by all peripheral mononuclear cells in sensitized individuals.

Anti-ICAM suppresses the proliferative response of human peripheral blood mononuclear cells in systems known to require cell-cell adhesion
The ability of the -1 monoclonal antibody R6-5-D6 was tested.

Peripheral blood mononuclear cells were transferred to Ficoll-Paqu
a, Pharmacia) Gradient purified as recommended by the manufacturer. After collecting the interface, the cells were washed three times with RPMI1640 medium and placed in a flat bottom 96-well microtiter plate.
10 ⁶ cells / m 2 in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine and gentamicin (50 μg / m)
At a concentration of 1.

Any of antigen, T-cell mitogen and concanavalin A (0.25 μg / m); T-cell binding antibody OKT ₃ (0.001
μg / m); keyhole limpet hemocyanin (10 g / m
) Or tetanus toxoid (1: 100 dilution from source) in the presence or absence of anti-ICAM-1 antibody (R6-5-D6; final concentration 5 g / m) in cells cultured as described above. Added. Cells were treated for 3.5 days (concanavalin A test), 2.5 days (OKT ₃ test), or 5.5 days before the end of the assay.
Cultured on day (keyhole limpet hemocyanin and tetanus toxoid test). 18 hours before the end of the assay,
2.5 μCi of ³ H-thymidine was added to the culture. Cell proliferation was assayed by measuring thymidine incorporation into DNA by peripheral blood mononuclear cells. Incorporated thymidine was collected and counted in a liquid scintillation counter [Merluzzi et al ., J. Immunol . 139 : 166-168 (1987)]. The results of these experiments are shown in FIG. 16 (concanavalin A test),
The results are shown in FIG. 17 (OKT ₃ test), FIG. 18 (keyhole limpet hemocyanin test), and FIG. 19 (tetanus toxoid test).

The anti-ICAM-1 antibody has a proliferative response to non-specific T-cell mitogens in mononuclear cells, ConA; non-specific T-cell associated antigen, OKT-3; and specific antigens, keyhole limpet hemocyanin and tetanus toxoid. It turned out to be suppressed. Inhibition by anti-ICAM-1 antibody is comparable to the inhibitory effect of anti-LFA-1 antibody, with ICAM-1 being a functional ligand of LFA-1 and antagonist of ICAM-1 inhibiting specific defense system responses. Suggests that it will.

Example 27 Effect of Anti-ICAM-1 on Mixed Lymphocyte Response As mentioned above, ICAM-1 is required for effective cell interactions during the immune response mediated by LFA-1-dependent cell adhesion . Induction of ICAM-1 during an immune response or inflammatory disease allows leukocytes to interact with each other or with endothelial cells.

When lymphocytes from two unrelated individuals are cultured in the presence of each other, blast transformation and lymphocyte cell proliferation are observed. This response of the leukocytes of one population to the presence of leukocytes of the second population is a mixed lymphocyte reaction (MLR)
And is analogous to the response of lymphocytes to the addition of mitogens [ Immunology the Science of Se
lf-Nonself Discrimination , Klein, J. John Wiley & S
one company, NY (1982), pp453-458].

The effect of anti-ICAM monoclonal antibody on human MLR was tested. These experiments were performed as follows. Peripheral blood was collected by venipuncture from normal healthy donors. Collect the blood in heparinized tubes and allow Punk's
It was diluted 1: 1 with G (GIBCO) balanced salt solution (BSS). The blood mixture (20 m) was layered on a 15 m Ficoll / Hypaque density gradient (Pharmacia, density 1.078, room temperature) and
Centrifuged at 0Xg for 20 minutes. The interface was then collected and washed three times in Punk's G. Count cells on a hemacytometer,
0.5% gentamicin, 1 mM L-glutamine (GIBCO
) And 5% heat-inactivated (56 ° C, 30 minutes) human AB serum (Flow Laboratories) in RPMI-1640 medium (GIBCO) (hereinafter referred to as RPMI medium).

Mouse anti-ICAM-1 (R6-5-D6) was used in this experiment. All monoclonal antibodies (prepared from ascites fluid by Jackson Immunoresearch Laboratories, Boston, MA) were used as purified IgG preparations. Peripheral blood single cells (PBMC) were 6.25 × 10 ⁵ cells / m in a Linbro round bottom microtiter plate (# 76-013-06)
In the medium. Stimulator cells from different donors were irradiated at 1000R and cultured at the same concentration as the responder cells. The total volume per culture was 0.2 m.
Controls included responder cells alone and stimulator cells alone. Culture plates at 37 ° C for 5
% CO ₂ - was incubated in a humidified air atmosphere for 5 days. Tritiated thymidine to each well 0.5 pCi ⁽³ HT)
Last 18 cultures (New England Nuclair)
Time pulsed. In some cases, two-way ML
R performed. The protocol was the same except that the second donor cells were not inactivated by irradiation.

Cells were harvested on glass fiber filters using an automated multiple sample harvester (Skatron, Norway) and rinsed with water and methanol. Dry the filter in the oven and place it in an aquasol with Begman (LS-38).
01) Counted with a liquid scintillation counter. Results are shown as mean CPM ± SEM for six individual cultures.

Table 15 shows that the purified anti-ICAM-1 monoclonal antibody was 20 ng / m2.
MLR in a dose-dependent manner with significant suppression apparent in
Was suppressed. Purified mouse IgG showed little or no inhibitory effect. Inhibition of MLR by anti-ICAM-1 monoclonal antibody occurs when the antibody is added within the first 24 hours of culture (Table 16) ^a Responder cells (6.25 × 10 ⁵ / m) ^b Stimulator cells (6.25 × 10 ⁵ / m, irradiation at 1000R) ^c Purified monoclonal antibody against ICAM-1 at final concentration (μg / m) (R6-5 -D6) or purified mouse IgG
(MIgG) ^d Mean ± SEM of 5-6 cultures, numbers in parentheses are
Shows% suppression of MLR.

^a Responder cells (6.25 × 10 ⁵ / m) ^b Stimulator cells (6.25 × 10 ⁵ / m) ^c Purified monoclonal antibody against culture medium or ICAM-1 (R6-5-D6), 10 μg / m at day 0 Added at 24 hour intervals.

If the mean value ± standard error ^e nd = ^f% inhibition summary without measurement of ^d 4 to 6 pieces of culture, ability to inhibit MLR of antibodies against ICAM-1 is treated ICAM-1 monoclonal antibodies in acute transplant rejection It shows that it has practical use. ICAM-1 monoclonal antibodies also have therapeutic utility in related immune-mediated irregularities that depend on LFA-1 / ICAM-1 regulatory cell-cell interactions.

The above experiments show that the addition of a monoclonal antibody to ICAM-1 suppresses the mixed lymphocyte reaction (MLR) when added during the first 24 hours of the reaction. Furthermore, ICAM-1 improves the condition of human peripheral blood mononuclear cells in in vitro culture.

Furthermore, ICAM-1 was found not to be expressed on quiescent human peripheral blood lymphocytes or mononuclear cells. ICAM
-1 is conditioned by using conventional flow cytometric analysis on mononuclear cells, either monocultured cells or co-cultured with unrelated donor cells in a mixed lymphocyte reaction. This improved status of ICAM-1 on mononuclear cells can be used as an indicator of inflammation, especially when ICAM-1 is expressed on fresh human mononuclear cells with acute or chronic inflammation.

Specificity of ICAM-1 for activated mononuclear cells and ICAM
The ability of antibodies to MLR-1 to suppress the MLR indicates that ICAM-1 monoclonal antibodies may have diagnostic and therapeutic potential in acute transplant rejection and related immune-mediated irregularities requiring cell-cell interactions ing.

Example 28 Synergistic Effect of Mixed Administration of Anti-ICAM-1 and Anti-LFA-1 Antibodies As shown in Example 27, MLR is suppressed by anti-ICAM-1 antibody. MLR can also be suppressed by anti-LFA-1 antibodies. To determine if the combined administration of anti-ICAM-1 and anti-LFA-1 antibodies had an enhanced or synergistic effect, an MLR assay (performed as described in Example 27) was performed on the two antibodies. In the presence of various concentrations of

This MLR assay showed that the combination of anti-ICAM-1 + anti-LFA-1 was significantly more effective at suppressing the MLR response at concentrations where the antibody alone did not dramatically suppress the MLR (Table 17). This result indicates that treatments involving co-administration of an anti-ICAM-1 antibody (or fragment thereof) and an anti-LFA-1 antibody (or fragment thereof) have the ability to provide improved anti-inflammatory treatment. Such improved therapy allows for the administration of lower antibody doses than other therapeutically effective methods, and is important in cases where high concentrations of individual antibodies elicit anti-idiotypic responses Is shown.

Example 29 Additional effects of sub-optimal doses of anti-ICAM-1 and other immunosuppressants in MLR at sub-optimal doses As shown in Example 28, MLR is composed of anti-ICAM-1 antibody and anti-LFA
-1 suppressed by a combination of antibodies. MLR assay to see if co-administration of anti-ICAM-1 with other immunosuppressants (such as dexamethasone, azethiopyrine, cyclosporin A or steroids (such as prednisone, etc.) also has improved effects. Is lower than the optimal concentration of R6-5-D6 in combination with other immunosuppressants (i.e., lower than the optimal concentration for administering the agent alone to a subject), as per the protocol of Example 27. Concentration).

The data show that the inhibitory effect of R6-5-D6 is at least added to that of suboptimal doses of dexamethasone (Table 18), azethiopyrine (Table 19) and cyclosporin A (Table 20). I have. This indicates that anti-ICAM-1
Can be effective in reducing the required dosage of known immunosuppressants, ie, in reducing their toxic side effects. To obtain such immunosuppression using an anti-ICAM-1 antibody (or fragment thereof), the antibody (or fragment thereof) may be combined with a single additional immunosuppressant or two or more additional immunosuppressants. It is possible to administer the combination.

Example 30 Anti-IC in inhibiting rejection of transplanted allogeneic organs
Anti-ICAM in suppressing the rejection of allogeneic transplanted organs
Allograft kidney was transplanted in cynomolgus monkeys according to the method of Cosimi et al. [Transplant. Proc. Modified using ketamine.

That is, kidney transplantation was performed essentially as follows. Atypical renal allografts were performed on 3-5 kg cynomolgus monkeys after induction of anesthesia with barium and ketamine essentially as described by Marquet [Marquet et al., Medical.
Primatology, Part II, Basel, Karger, p. 125 (197
2)]. End-to-lateral anastpmoses of the donor kidney on aortic or vena cava patch 7-0
Constructed using Prolene suture. The donor ureter was spatula-treated and implanted in a bladder by an external method (Taguchi, Y. et al., Dausset et al., Advan
ces in Transplantation, Baltimore, Williams
And Wilkins, p393 (1968)]. Kidney function was assessed by weekly or biweekly serum creatinine measurements. In addition, frequent allograft biopsies were taken for histopathology and complete dissections were performed on all dead recipients. For most receptors, bilateral nephrectomy was performed at the time of transplantation, and subsequent uremic death was considered the endpoint of allograft survival. For some receptors, unilateral live nephrectomy and contralateral ureteral ligation were performed at the time of transplantation.
When allograft rejection occurred, the ligature on the same ureter was removed and a machine was obtained to continue normal kidney function regeneration and immunological monitoring of recipient animals.

Monoclonal antibody R6-5-D6 was administered daily for 12 days, starting with a dose of 1-2 mg / kg / day from 2 days prior to emigration. Serum levels of creatinine were tested synchronously to monitor rejection. Anti-ICAM against allogeneic kidney immune system rejection
The effects of one antibody are shown in Table 21.

^a monkeys were given R6-5-D6 daily for 2 to 12 days before transplantation.

^bThe cause of death was unknown. There was evidence of a potential malaria.

^c Renal infarction death ^d Still alive on August 15, 1988 The above results indicate that R6-5-D6 is effective in extending the lifespan of monkeys that received allogeneic transplantation.

Example 31 Effect of Anti-ICAM-1 Antibody on Inhibiting Acute Rejection of Transplanted Organs To demonstrate that anti-ICAM-1 antibodies are effective in an acute model of transplant rejection, Or tested in an acute renal rejection model. In this model, monkey kidneys were transplanted (using the protocol of Example 30) and 15 mg / kg cyclosporin A (CyA) was administered intramuscularly until stable renal function was obtained peri-tissue. The CyA dose was then reduced every 2.5 weeks in 2.5 mg / kg increments until rejection occurred as indicated by elevated blood creatinine levels. At this point, R6-5-D6 was administered for 10 days and survival time was monitored. It is important to note that in this protocol, the dose of CyA remains suboptimal as it does not change as soon as the acute rejection condition occurs. In this model, histological controls without antibody assistance (N = 5) were 5 to 5 days after the onset of rejection.
Survive for 14 days. To date, six animals have been tested with R6-5-D6 in this protocol (Table 22).
Two of these animals are still alive (M12: 31 days and M5: 47 days after administration of R6-5-D6). Two animals are R
Living 38 and 55 days after the start of 6-5-D6 treatment, two animals died of causes other than acute rejection (one died of CyA toxicity and one underwent R6-5-D6 under anesthesia). Died during administration). This model more closely approximates the clinical situation in which R6-5-D6 was initially used.

^a Monkeys had 1-2 mg / kg of R6-5 daily for 10 days after the start of rejection
D6 was administered.

except ^b creatinine level CyA doses of increased R6-5-D6 therapy and started day ^c 5 animals as a result of the reduction was not used assistance therapy was tested using the aforementioned treatment protocol. The number of days of survival / post-treatment indicates the number of days immediately after creatinine levels began to rise.

^d Died during anesthesia, creatinine level was low.

^e Death due to CyA toxicity. Creatinine levels were low.

^f Still alive on August 15, 1988.

Example 32 Gene Construction and Expression of Truncated Derivatives of ICAM-1 In its native state, ICAM-1 is a cell membrane-associated protein containing the extramembrane domain of five immunoglobulin-like domains and the cytoplasmic domain. It was desirable to construct functional derivatives of ICAM-1 that lack transmembrane domains and / or cytoplasmic domains in that they can express solubles secreted from ICAM-1. These functional derivatives were constructed by oligonucleotide-related mutagenesis of the ICAM-1 gene and subsequent expression in monkey cells after introduction with the mutated gene.

Amino acid substitution and / or truncation (Truncate
d) Mutations in the ICAM-1 gene that give rise to the derivative are described in Kunkel,
[Proc. Natl. Acad. Sci. (U.
SA) 82: 488-492 (1985)]. The ICAM-1 cDNA prepared as described above was digested with the restriction endonucleases Sal1 and K.
digested with pm1, and the resulting 1.8 kb DNA fragment was subcloned into a plasmid vector CDM8 (Seed, B. et al., P.
roc.Natl.Acad.Sci. (USA) 84: 3364-3369 (198
7)]. Next, the dut ⁻ and ung ⁻ strains of E. coli (BW313 / P3) were
Transformation with the above construct, designated 1.8C. A single strand uracil containing template was rescued by infecting the transformants with helper phage R408 (Stratagene R). The mutant ICAM-1 cDNA is then produced by initiating a second strand synthesis with an oligonucleotide having a mismatched base and subsequent transformation of the ung ⁺ host ( MC1061 / P3) with the resulting heteroduplex. Was. Mutants were isolated by screening for newly created endonuclease restriction sites into which the mutant oligonucleotide was introduced. The mutant ICAM-1 protein is prepared by the standard DEAE-dextran method [Selden, RF et al., Current Protocols in Molecular
Biology (edited by Ausebel, FM, etc.), 9.2.1-9.2.6 (198
7)] using the above mutant D in the eukaryotic expression vector CDM8.
Expressed by transfer of Cos-7 cells with NA.

A truncated functional derivative of ICAM-1 that lacks the transmembrane domain and the cytoplasmic domain but contains all five immunoglobulin-like domains was prepared. 30bp mutant oligonucleotide (CTC TCC CCC CGG
Positions 452 and 453 using TTC TAG ATT GTC ATC ATC)
The amino acid tyrosine (Y) and glutamic acid (E) codons were transformed into phenylalanine (F) and translation stop codon (TAG), respectively. Mutant was isolated by its characteristic Xba 1 restriction site, and displays Y ⁴⁵² E / F, and TAG.

To express the mutant proteins, three mutant subclones (# 2, # 7 and # 8) were transferred into COS cells. Three of the transfections with these three mutant subclones
One day later, culture supernatants and cell lysates were analyzed by immunoprecipitation with anti-ICAM-1 monoclonal antibody RR1 / 1 and SDS-PAGE. ICAM-1 precipitated from the culture supernatant of cells transfected with mutant subclones # 2 and # 8, but not from detergent lysates of these cells. The molecular weight of ICAM-1 found in the culture supernatant was reduced by about 6 Kd compared to the membrane of ICAM-1, which is consistent with the size expected from the mutant DNA. That is, this functional derivative of ICAM-1 is secreted as a soluble protein. In contrast, IC
AM-1 did not immunoprecipitate from the culture supernatant of cells transfected with control native ICAM-1, indicating that the ICAM-1 membrane was not expressed from COS cells. Furthermore, ICAM-
1 did not immunoprecipitate from culture supernatants or cell lysates from negative control mock-transfected cells.

The truncated ICAM-1 secreted from the transferred cells was replaced with ICAM-1.
Purified by immunoaffinity chromatography with a specific antibody (R6-5-D6) and tested for functional activity in a cell binding assay. After purification in the presence of the detergent octylglucoside, the preparation containing native ICAM-1 or truncated secretor was diluted to a final concentration of 0.25 octylglucoside (subcritical micellar concentration of detergent). . These preparations of ICAM-1 were bound to the surface of a plastic 96-well plate (Nunc) to prepare solid phase bound ICAM-1. After washing out unbound, about 75-80% and about 83-85% of SKW-3 cells containing LFA-1 on the surface are specific for the native and truncated forms of ICAM-1, respectively. Bound. These data demonstrate that secreted truncated soluble ICAM-1 functional derivatives retain both immunological reactivity and the ability to mediate ICAM-1-dependent adhesion, a characteristic of native ICAM-1. Is shown.

A functional derivative of ICAM-1 lacking only the cytoplasmic domain was prepared in a similar manner. The 25 bp oligonucleotide (TC AGC ACG TAC CTC TAG AAC CGC CA) was used to change the codon for amino acid 476 (Y) to a TAG translation stop codon. This mutant was designated as ^Y476 / TAG. Immunoprecipitation analysis of Cos cells transfected with this mutant and SDS-PAGE were negative IC
A membrane-bound form of ICAM-1 having a molecular weight about 3 Kd smaller than AM-1 was detected. Indirect immunofluorescence analysis of the mutant-transfected Cos cells revealed that native ICAM expressed on LPS-stimulated human endothelial cells.
-1 showed the same point-like stained image. In addition, the mutant DN
Cells transfected with A bound specifically to purified LFA-1 on plastic surfaces in a manner similar to Cos cells transfected with native ICAM-1 DNA (Table 23).

Example 33 Mapping of ICAM-1 functional domain Studies of ICAM-1 have found that the molecule has seven domains. Five of these domains are extracellular (domain 5 is closest to the cell surface and domain 1 is farthest from the cell surface), one domain is transmembrane domain and one domain is cytoplasmic ( That is, it exists in the cell). Which domain is IC
To determine if it contributes to the ability of AM-1 to bind LFA-1, an epitope mapping (mapping) test was used. To perform such tests, different deletion mutants were created and characterized for their ability to bind LFA-1. Separately, tests were performed using anti-ICAM antibodies that are known to interfere with the ability of ICAM-1 to bind to LFA-1. Suitable examples of such antibodies include RR1 / 1 (Rothlein, R., etc.
J. Immunol. 137: 1270-1274 (1986)], R6.5 (Springa
r, TA et al., U.S. Patent Application No. 07 / 250,446), LB-2 [Clar
k, EA, etc., Leukocyte Typing I (edited by A. Bernard, etc.), S
pringer-Verlag, 339-346 (1984)], or CL203
[Staunton, DE et al., Cell. 56: 849-853 (1989)].

A deletion mutant of ICAM-1 can be prepared by any of a variety of methods. However, such variants may be obtained by site-directed mutagenesis or by other recombination means (such as by constructing an ICAM-1 expressing gene sequence that lacks sequences encoding particular protein regions). Preferably, it is produced. Suitable procedures for producing such variants are well known in the art. Using such a procedure, three ICAM-1 deletion mutants were prepared. First
Variant lacks amino acid residues F185 to P284 (ie,
Domain 3 missing). The second variant is amino acid residue P284
~ R451 is missing (i.e., domains 4 and 5 are missing). The third mutant lacks the amino acid residue after Y476 (ie, lacks the cytoplasmic domain). The results of such tests indicate that domains 1, 2 and 3 are primarily involved in ICAM-1 interaction with anti-ICAM-1 antibodies and LFA-1.

Example 34 Effect of ICAM-1 Mutation on LFA-1 Binding The ability of ICAM-1 to react and bind to LFA-1 is ICAM-1
It is mediated by the ICAM-1 amino acid residues present in domain 1 of the molecule (FIGS. 8, 9 and 10). However, such a reaction is facilitated by the contribution of amino acids present in domains 2 and 3 of ICAM-1. That is,
Among the preferred functional derivatives of the present invention are soluble fragments of the ICAM-1 molecule containing domains 1, 2 and 3 of ICAM-1. More preferred is ICAM-1
Is a soluble fragment of the ICAM-1 molecule containing domains 1 and 2. Most preferred is a soluble fragment of ICAM-1 containing domain 1 of ICAM-1. Some amino acid residues in the first ICAM-1 domain are I
It is included in the reaction between CAM-1 and LFA-1. Substitution of these amino acids with other amino acids alters the ability of ICAM-1 to bind to LFA-1. These amino acid residues and their substitutions are shown in FIG. Figure 25 shows the obtained mutant
9 shows the effect of such a mutation on the ability of the ICAM-1 molecule to bind LFA-1. In FIGS. 23-25, each residue is described in terms of the one letter code for the amino acid, followed by the IC
The position of that residue in the AM-1 molecule is indicated. Thus, for example, "E90" refers to the glutamic acid residue at position 90 of ICAM-1. Similarly, “E90V” indicates a dipeptide consisting of a glutamic acid residue at position 90 and a valine residue at position 91. The replacement sequence is shown to the right of the slash ("/") mark.
V4, R13, Q27, Q58 and D60S61 residues of ICAM-1 are LFA-
Included in one bond.

Substitution of these amino acids alters the ability of ICAM-1 to bind to LFA-1. For example, replacement of V4 by G is LFA-
Substitution of E13 for the R13 residue of ICAM-1 results in the formation of a mutant ICAM-1 molecule that binds less to 1 (FIG. 25). (Fig. 25). Q58 of ICAM-1
Substitution of the residue with an H results in a mutant molecule having substantially the ability to bind LFA-1 (FIG. 25). ICAM-1
Substitution of the D60S residue with KL results in a mutant molecule with substantially less ability to bind LFA-1 (FIG. 25).

A glycosylation site in the second domain is also involved in LFA-1 binding (FIG. 23). By K of N103 or A155
Replacement of N by SV results in the formation of a mutant ICAM-1 molecule that cannot substantially bind LFA-1. In contrast, replacement of the glycosylation site N 175 by A results in the L of the mutant ICAM-1
It did not appear to substantially affect FA-1 binding capacity.

Mutations in the 31st CAM-1 domain did not appear to appreciably alter ICAM-1-LFA-1 binding (24th CAM-1).
Figure).

Example 35 A multimeric form of ICAM-1 with increased biological half-affinity and clearance ability A quinola molecule is constructed in which domains 1 and 2 of ICAM-1 are linked to the hinge region of its immunoglobulin long chain. Preferred constructs link the C-terminus of ICAM-1 domain 2 to a segment of the immunoglobulin long chain just N-terminal to the hinge region to allow for the segment flexibility conferred by the hinge region. ICAM-1 domain 1
And 2 will thus replace the Fab fragment of the antibody. Binding of IgGs to long chains and generation of animal cells will result in the production of chimeric molecules. Generation of molecules containing long chains from IgA or IgM resulted in 2-12 ICAM-1
It will result in the production of higher multimeric molecules containing the molecule. Co-expression of the long chain gene in animal cells producing the ICAM-1 long chain chimeric molecule is based on the expression of 4-6 IgA molecules containing ICAM-1 molecules and about 10 IgM molecules containing ICAM-1 molecules. It will provide a suitable assembly of IgA and IgM multimers that will primarily provide the case. These chimeric molecules have several advantages. First, the Ig molecules are designed to stay longer in the circulatory system, which can improve biological half-life.

Furthermore, the multimeric nature of these engineered molecules may, depending on the therapeutic context, allow these molecules to react with rhinovirus and cell surface LFA-1 with higher avidity, thus giving an effective dose. Significantly reduces the amount of recombinant protein required. IgA and IgM are highly glycosylated molecules normally present in secretions of mucosal sites such as in the nose. Its high hydrophilicity helps to retain microbes and viruses that bind in the mucosa, prevents binding to cells, and prevents crossing of the epithelial cell membrane barrier. That is, these molecules have an increased therapeutic effect. IgM and especially IgA are stable in mucosal environment and ICAM
-1 Increase the stability of the construct. Such ICAM-1
When a functional derivative is administered into the bloodstream, it also increases the biological half-life. IgA does not fix complement and therefore would be ideal in applications where it is harmful.
If a heavy chain chimera of IgG is desired, binding to complement and F
It would be possible to mutate the region involved during the reaction with the c receptor.

Example 36 Expression of ICAM-1 Mutant Oligonucleotide Specific Mutagenesis The coding region of the ICAM-1 cDNA in the 1.8 Kb Sal1-Kpm1 fragment was subcloned into the expression vector CDMB (Seed, B. et al., Proc. Natl. Acad. .Sci. (USA) 84: 3365
-3369 (1987)]. A method of Kunkel, T. (Proc. Natl. Acad.
Sci. (USA) 82: 488-492 (1985)] and Staunton.
D. et al. (Staunton DE et al., Cell 52: 925-933 (198
8)], this construct (pCD1.8) was used to prepare a single-stranded uracil-containing template for use in oligonucleotide-directed mutagenesis.

Briefly, E. coli strain XS127 was transformed with pCD1.8.
A single colony was harvested from a 1 m luria broth (L) containing 13 μg / m ampicillin and 8 μg / m tetracycline.
B) Grow to near saturation in medium (Difco). 100μ
Culture with R408 helper phage (Strategene)
The cells were infected at a multiplicity of infection (MOI) of 10 and 10 m of LB medium containing ampicillin and tetracycline was added thereto at 37 ° C. for 16 hours. Centrifuge for 1 minute at 10,000 rpm and 0.2 ml of supernatant
After 2 μm filtration, the phage suspension was infected with E. coli BW313 / P3, which was then spread on LB agar (Difco) plates supplemented with ampicillin and tetracycline. Colonies were picked, grown to near saturation in 1 m LB medium containing ampicillin and tetracycline, and infected with helper phage at MOI10. The culture volume was then increased to 250
m and the cells were cultured overnight. Single strand D
NA was isolated by standard phage extraction.

The mutant oligonucleotide was phosphorylated and used in a second strand synthesis reaction with the pCD1.8 template [Staunton D. et al.
E. et al., Cell 52: 925-933 (1988)].

Transfer 10% of COS cells to 50% confluency in 16-24 hours.
m of tissue culture plates. Then, COS
Wash cells once with TBS and add 10% Nu serum (Collaborative
5 μg / m chloroquine, 3 μg
Of the mutant plasmid and 200 μg / m of DEAE-dextran sulfate in 4 m of RPMI for 4 hours. The cells are then washed with 10% DMSO / PBS then PBS,
The cells were cultured in a culture medium for 16 hours. The culture medium is replaced with fresh medium, and at 48 hours, the post-transfected COS cells are replaced with trypsin / EDTA
(Gibco) treatment and resuspended in 2, 10 cm plates for HRV binding and 24-well tissue culture plates.
At 72 hours, cells were harvested from 10 cm plates with 5 mM EDTA / HBSS and processed for adhesion to LFA-1 coated plastic and immunofluorescence analysis.

LFA-1 and HRV binding LFA-1 was purified from SKW-3 on TS2 / 4LFA-1 mAb Sepharose by immunoaffinity chromatography and 2 m
PH in the presence of M MgCl ₂ and 1% octyl glycoside
Eluted at 11.5. By diluting LFA-1 (10 μg / 200 μ / 6 cm plate) in a microbiological Petri dish with 0.1% octylglucoside in PBS containing ₂ mM MgCl ₂ (phosphate buffered saline) and incubating overnight at 4 ° C. Combined. Each plate was blocked with 1% BSA (bovine serum albumin) and PBS containing ₂ mM MgCl ₂ , 0.2% BSA, 0.025% azide, and 50 μg / m gentamicin
Saved in.

⁵¹ Cr-labeled COS cells in PBS (buffer) containing 5% FCS (fetal calf serum), 2 mM MgCl ₂ , 0.025% azide in RFA-1 coated microtiter plates at 5 μg / m RRI / 1 and In the presence or absence of R6.5
Incubated for 1 hour at 25 ° C. Non-adherent cells were removed by washing three times with buffer. Adherent cells were eluted to 10 mM by the addition of EDTA and γ-calculated.

RESULTS Anti-ICAM-like RR1 / 1, R6.5, LB-2, or CL203
One antibody has been identified. If these antibodies can suppress ICAM-1 function, they must be able to bind to specific sites on the ICAM-1 molecule, which are also important for ICAM-1 function. That is, by preparing the above-mentioned ICAM-1 deletion mutant and measuring the extent to which the above-mentioned anti-ICAM-1 antibody can bind to the above-mentioned deletion body, it is determined whether the deletion domain is functionally important. Can be.

ICAM-1 is a complex membrane protein whose extracellular domain is predicted to consist of five 1g-like C-domains. To identify the domains involved in binding LFA-1, domains 3, and domains 4 and 5 (less than carboxyl) were deleted by oligonucleotide-directed mutagenesis and tested functionally after expression in COS cells. did. In addition, the deletion of the entire cytoplasmic domain confirmed its potential effect on ICAM-1 response. As expected, the cytoplasmic domain deletion, Y476 / ^* was RR1 / 1, R6.5, LB-2 and CL20
The lack of domain 3 F184-R451 resulted in a decrease and loss of CL203 reactivity, while showing no loss of 3 reactivity (FIG. 20). That is, although the CL203 epitope appears to be present in domain 4, RR1 / 1, R6.5 and LB-
2 appears to be in two amino-terminal domains.

All three deletion mutants exhibited wild-type levels of adhesion to LFA-1 (FIG. 21). Amino acid substitutions at predicted β-turns in domains 1, 2 and 3 were also prepared and COS
Functionality was tested after expression in cells. The R6.5 epitope is localized to the sequence E111GGA of domain 2 and
RR1 / 1 and LB-2 are both domain 13 R13
(Fig. 22). Furthermore, RR1 / 1 binding was reduced by mutations in sequence D71GOS. Mutations that reduce the N-linked glycosylation sites of N103 and N163 are RR1 / 1, R6.
5 and reduced LB-2, LFA-1 HRV binding. These mutations appear to be engineered to generate ICAM-1 dimers.

Other mutations in domain 2 or 3 did not result in altered LFA-1 adhesion (FIGS. 23 and 24). Both domain 13 amino acids, R13 and D60, are involved by binding to LFA-1 (FIG. 25).

Thus, LFA-1 and HRV binding appear to be a function of the sub-amino Ig-like domain of ICAM-1. Figure 26 shows ICAM
1 shows the sequence of the domain less than -1.

Although the invention has been described with respect to particular embodiments thereof, it will be understood that different modifications are possible and that the present application generally is in accordance with the principles of the invention and within the art to which the invention pertains. Inclusive of all variations, uses, or adaptations, including circumvention from the description herein, as included in known or conventional practice in US and applicable to the essential features as claimed.

──────────────────────────────────────────────────の Continuation of front page (51) Int.Cl. ⁶ Identification symbol FI C07K 16/18 C12P 21/02 C C12N 15/09 ZNA C12N 15/00 ZNAA C12P 21/02 A61K 37/02 (72) Inventor Merlin Stephen Dee United States Connecticut 06810 Danbury Terra Hot Road 18 (72) Inventor Dustin Michael Eller United States Massachusetts 02215 Boston 23 Park Drive 231 (58) Fields studied (Int. Cl. ⁶ , DB name) C07K 14 / 78 C07K 16/18 C12N 15/06

Claims (16)

(57) [Claims] (1) at least amino acid residue 45 of native ICAM-1;
A soluble functional derivative of ICAM-1, which differs from ICAM-1 in that the transmembrane domains 4 to 477 are missing, comprising domain 1 of amino acid residues 1 to 88, Such functional derivatives that retain the ability to mediate the immunological reactivity and ICAM-1-dependent adhesion characteristic of ICAM-1 and are soluble under physiological conditions. 2. The soluble functional derivative of ICAM-1 according to claim 1, comprising at least one altered amino acid residue and capable of binding to a CD18 family member molecule. 3. ICAM-1 further comprising amino acid residues 478 to 505.
3. The ICAM of claim 1 or 2 lacking the cytoplasmic domain of
-1 soluble functional derivative. 4. An amino acid residue after amino acid residue 88 of domain 1 or after amino acid residue 185 of domain 2 or after amino acid residue 283 of domain 3 Item 4. A soluble functional derivative of ICAM-1 according to any one of Items 1 to 3. 5. The ICAM- according to any one of claims 1 to 4, wherein
Characterized by containing a soluble functional derivative of 1
A pharmaceutical composition for treating inflammation resulting from a specific or non-specific defense system response. 6. ICAM- according to any one of claims 1 to 4.
Characterized by containing a soluble functional derivative of 1
A pharmaceutical composition for inhibiting the transfer of hematopoietic tumor cells that requires a functional member of the LFA-1 group for transfer. 7. The ICAM- according to any one of claims 1 to 4, wherein
A pharmaceutical composition for suppressing the growth of LFA-1-expressing tumor cells, comprising a soluble functional derivative derived from 1 toxin. 8. An immunosuppressant selected from dexamethasone, azathiopyrine and cyclosporin A, and an immunosuppressant selected from the group consisting of:
A pharmaceutical composition for treating inflammation resulting from a response of a specific defense system, comprising a soluble functional derivative of ICAM-1 according to any one of the above. 9. An antibody capable of binding to LFA-1, a functional derivative of the antibody which is capable of binding to LFA-1, and a second drug selected from non-immunoglobulin antagonists of LFA-1. A pharmaceutical composition for treating inflammation resulting from a response of a specific defense system, comprising a soluble functional derivative of ICAM-1 according to any one of claims 1 to 4. 10. The pharmaceutical composition according to claim 5, further comprising a carrier or an excipient. 11. The ICAM according to claim 1, wherein:
A recombinant DNA molecule capable of expressing a soluble functional derivative of -1. 12. An ICAM according to any one of claims 1 to 4.
Culturing a host microorganism transformed with a recombinant vector encoding a soluble functional derivative of -1 under conditions in which the derivative is expressed and secreted into the medium, and then isolating the derivative IC, including
A method for producing a soluble functional derivative of AM-1. 13. The ICAM according to claim 1, which is a fragment of ICAM-1 capable of binding to a molecule present on the surface of a lymphocyte.
-1 soluble functional derivative. 14. The soluble functional derivative of ICAM-1 according to claim 1, which is a mutant of ICAM-1 capable of binding to a molecule present on the surface of a lymphocyte. 15. The ICAM-1 according to claim 1, which is an analog of ICAM-1 capable of binding to a molecule present on the surface of a lymphocyte.
A soluble functional derivative of 16. The ICAM-1 according to claim 1, which is a chemical derivative of ICAM-1 capable of binding to a molecule present on the surface of a lymphocyte.
1. A soluble functional derivative of 1.