DD297839A5 - ALPHA SUB-UNIT OF THE LFA-L-LEUKOZYTADHAESION RECEPTOR - Google Patents

Abstract

The invention relates to a process for the preparation of an LFA-1 alpha subunit or its functional derivative, substantially free of natural contaminants, characterized in that a suitable host organism with an expression vector which as a coding sequence replicon and control sequence for expression in Prokaryotes and eukaryotes for the desired polypeptide at an appropriate site, transformed, or the LFA-1 alpha subunit or its functional derivative is chemically synthesized, and the desired polypeptide is isolated from the resulting transformants or reaction mixture. The LFA-1 alpha subunit is involved in the process by which the cell recognizes and migrates sites of inflammation as well as attacks on cellular substrates. {Alpha subunit of the leukocytic adhesion receptor; LFA-1 production; pharmaceuticals}

Description

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Field of application of the invention

The present invention relates to a method for producing the leukocyte adhesion receptor LFA-1. The invention also relates to the cloning of DNA sequences encoding the alpha subunit of this molecule. The invention has been partially realized with the support of the government. D ^! e Government has certain rights to this invention.

Characteristic of the known state of the art

The immune system is responsible for the protection of animals from foreign invaders, such as bacteria, viruses, etc. An excellent review of the defense system is provided by Eisen, H.W. given in: Microbiology (Microbiology), 3rd ed., Harper & Row, Philadelphia, PA [1980], pp. 290 to 295 and 381 to 418). The ability of the immune system to protect an animal from foreign vasodilators is highly dependent on the presence and function of blood cells known as leukocytes. It has been found that the ability of leukocytes to provide this protection requires adherence of these cells to cellular and extracellular substrates.

For example, leukocytes must be able to bind to endothelial cells so that they can migrate from the circulation to sites of incipient inflammation. In addition, they must bind to antigen-presenting cells, so that a normal immune response can occur. They must also be able to bind to appropriate target cells so that lysis can be from virally infected (or tumor) cells. In addition, leukocytes must be able to bind to various activated proteins (for example, iC3b -the activated form of the human component of the

Complement) so that they are effective in phagocytosis and can eliminate microbial and cellular debris. Thus, leukocyte adhesion is a prerequisite for a normally functioning host defense system. The suppression of this defense system is desirable in such cases as transplantation, as the host "sees" the transplanted tissue as foreign and initiates an immune reaction to this tissue, which also contributes to the rejection of transplanted tissues and organs To enable understanding of leukocyte adhesion, either to enhance an animal's ability to fight infections, or to suppress the ability of an animal to reject transplanted tissue.

Recently, leukocyte surface molecules involved in mediating leukocyte adhesion have been identified using hybridoma technology. Briefly, monoclonal antibodies directed against monoclonal T cells (Davignon, D. et al., Proc. Natl. Acad. Sc. USA 78: 4535-4549 [1981]) and mouse spleen cells (Springer, T. et al., Eur Immunol.9: 301-306 [1979]), which bind to leukocyte surfaces and prevent the binding-related functions described above (Springer T. et al., Fed. Proc.44: 2660-2663 [1985]). The molecules recognized by these antibodies form a set of leukocyte adhesion receptors known as the "lymphocyte function-related antigen-1 family" (or the "LFA-1 family") of adhesion receptor molecules. The LFA-1 family of adhesion receptor molecules contains three highly related cell surface glycoproteins. It has been found that these glycoproteins mediate cell-cell interactions in inflammation. The glycoproteins were termed "LFA-1" (lymphocyte function-related antigen-D, "Mac-1" and "p 150.95.") While LFA-1 is found on the surfaces of most leukocytes (Springer, TA et al., Immunol. 68: 111-135 [1982]), Mac-1 and ρ 150.95 are found predominantly on macrophages, granulocytes and other large granular lymphocytes (Springer, TA, et al., Immunol. Rev. 68: 111-135 (1982); Keizer.G., et al., Eur.J. Immunol., 15: 1142-1147 [1985]).

The LFA-1 glycoprotein family is composed of heterodimers each containing an alpha subunit noncovalently assigned to a beta subunit. It has been found that the alpha 'subunits of the family differ from each other, and they are referred to as CD 11 a, CD 11 b and CD 11 с, respectively. The glycosylated alpha subunits have approximate molecular weights of 180.170 and 150 kd, respectively. In contrast, it was found that the beta subunit of the LFA-1 family of adhesion receptors is identical and has a molecular weight of 95 kd dhat (Sanchez-Madrid, F. et al., J. Exp. Med.158: 1785-1803 [1983]; , GD et al., Eur.J. Immunol., 15: 1142-1147 [1985]; Springer, T., Fed. Proc.44: 2660-2663 [1985]; Sanchez-Madrid, F. et al., J.Exper.Med .158: 581-602 (1983)).

Although the alpha subunits of the glycoproteins do not have the extensive homology of the beta subunits, a thorough analysis of the alpha subunits of glycoproteins has shown that there are substantial similarities between them. An overview of the similarities between alpha and beta subunits of the adhesion molecule · glycoprotein family is provided by Sanchez-Madrid, F. et al. a. (J. Exper. Mod. 158: 586-602 (1983); J. Exper. Med. 158: 1785-1803 [1983]; Miller, L.J., et al., J. Immunol. 138: 2381-2383 [1987]).

The importance of the LFA-1 family of receptors was first recognized in studies that demonstrated the ability of monoclonal antibodies (which were able to bind to the specific alpha subunits or to the common beta subunit) to adhere to adhesion-dependent leukocyte functions Sanchez-Madrid, F. et al., Proc. Natl. Acad. Sc. USA 79: 7489-7493 (1982); Beller, DI et al., J.Exper. Med. 156: 1000-1009 (1982)).

Recently, a group of individuals have been identified who are unable to express normal levels of any member of the LFA-1 adhesion protein family on their leukocytic surfaces. This disease has been termed "leukocytic adhesion deficiency" or "LAD" and is characterized by chronic and recurrent infections as well as other clinical symptoms (Anderson, DC et al., Fed. Proc.44: 2671-2677 (1985); Anderson, DC et al., J Infect. DIs. 152: 668-689 [1985]). Leukocytes from LAD patients have in vitro defects that are very similar to those observed when leukocytes from normal individuals were fought by antibodies specific for members of the LFA-1 family. It has been found that LAD patients are unable to produce a normal immune response. It has been found that this failure is due to an inability of the leukocytes of LAD patients, both cellular and extracellular. Substrates (Anderson, D.C., et al., Fed. Proc.44: 2671-2677 [1985]; Anderson, D.C. et al., J.Infect. Dis.152: 668-689 [1985]). These studies indicate that inflammatory responses are attenuated when leukocytes are unable to adhere normally to their cell surface due to the lack of functional adhesion molecules.

In summary, therefore, the ability of leukocytes to maintain the health and viability of an animal requires that they be able to adhere to other cells (e.g., endothelial cells) and proteins (eg, ІСЗЬ). This adherence has been found to cause contacts involving specific receptor molecules present on the leukocyte surface of leukocytes. It has been found that these cell surface receptor molecules are strongly related. People whose leukocytes lack these cell surface receptor molecules have chronic and recurrent infections as well as other clinical symptoms.

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

Object of the invention

The process according to the invention provides the pharmaceutically valuable alpha subunit of the leukocyte adhesion receptor LFA-1.

Explanation of the essence of the invention

It is an object of the present invention to find pharmaceutically valuable cell surface receptor molecules and to provide processes for their production.

According to the invention, a method for producing the alpha subunit of the LFA-1 receptor molecule by the use of the recombinant DNA technique is provided.

In particular, the method according to the invention relates to the cloning and expression of the alpha subunit of the LFA-1 receptor molecule.

In particular, the invention relates to a process for producing an LFA-1 alpha subunit or its functional derivatives which are substantially free of natural contaminants.

The invention further relates to the preparation of the abovementioned LFA-1. Alpha subunit or its functional derivative, which are additionally capable of forming a molecule present on the surface of a cell (such as ICAM-1 or the LFA-1). Beta subunit).

The invention also includes the LFA-1. Alpha subunit molecule mentioned above, wherein the molecule contains at least one polypeptide selected from the group consisting of:

а. P-P-R-A-G-R-H; H. G-V-D-V-Q-Rv-G-E-I-E; b. μμΤ-D-G-E-A; i. D-I-N-G-D-G-L-V-D-V; C. W-A-G-G-F-L; j. V-K-D-L-E-G-D-G-L-A; d. S-Q-V-Q-T-I-H; k. T-Y-L-S-G-L; Θ. R-H-G-G-L-S-P; I. Y-H-G-I-G-K; f. M-S-C-T-D-F-S; m. μΕ-G-T-Q-V-L-S-Q and G- R-L-L-S-R-A-L; n. P-S μΗ-Ν-Ι-Ρ.

The invention also includes a recombinant DNA molecule capable of expressing either the LFA-1 alpha subunit or its functional derivative.

The invention also relates to a method for obtaining the LFA-1 alpha subunit h of a substantially pure form, which consists of the steps:

(a) solubilization of the LFA-1 alpha subunit from the membranes of cells expressing the LFA-1 alpha subunit to yield a solubilized LFA-1 alpha subunit preparation;

(b) introducing the solubilized LFA-1 alpha subunit preparation into an affinity matrix, wherein the matrix contains immobilized antibody capable of binding the LFA-1 alpha subunit;

(c) allowing binding of the LFA-1 alpha subunit to the antibody of the affinity matrix;

(d) removal of any compound which is unable to bind to the antibody from the matrix and

(e) recovering the LFA-1 alpha subunit in a substantially pure form by eluting the LFA-1 alpha subunit from the matrix.

The invention also provides a method for controlling inflammation resulting from a reaction of the specific defense system (such as delayed hypersensitivity, rejection of tissue transplantation, rejection of organ transplants, autoimmune diseases such as lupus erythemotosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of Diabotis, Reynaud's syndrome, rheumatoid arthritis, etc.) in a mammal, which is to administer a sufficient amount of an anti-inflammatory agent to a subject in need of such treatment to suppress the inflammation; wherein the anti-inflammatory agent is selected from the group consisting of LFA-1 alpha subunit and LFA-1 alpha subunit functional derivative.

The invention further includes the above-mentioned method additionally providing co-administration of an agent selected from the group consisting of LFA-1 beta subunit and a functional derivative of LFA-1 beta subunit.

The invention also relates to a method for suppressing the metastasis of a harr Mopoietischen tumor cell, which needs a functional member of the LFA-1 family for walking, the method is a patient who needs such treatment, a sufficient amount of a anti-inflammatory agent to suppress metastasis, wherein the anti-inflammatory agent is selected from the group consisting of: LFA-1 alpha subunit and functional derivative of the LFA-1 alpha subunit.

The invention relates to a method of suppressing the growth of an LFA-1 alpha subunit-expressing tumor cell, which comprises administering to a patient in need of such treatment a sufficient amount of a growth suppressing toxin Toxin is selected from the group consisting of a toxin-derived LFA-1 alpha subunit and a toxin-derived functional derivative of a member of the LFA-1 alpha subunit.

The invention also relates to a method of diagnosing the presence and location of inflammation resulting from the response of the specific defense system in mammals suspected of having an inflammation consisting of:

(a) administer to the individual a composition containing a detectably labeled LFA-1 alpha subunit capable of identifying a cell expressing ICAM-1 or another natural ligand of LFA-1, and

(b) to detect the LFA-1 alpha subunit.

The invention also relates to a method of diagnosing the presence and location of inflammation resulting from the reaction of the specific defense system in mammals suspected of having an inflammation consisting of:

(a) incubating a subject tissue sample with a composition containing a detectably labeled LFA-1 alpha subunit capable of identifying a cell containing the ICAM-1 or other natural ligand of LFA-1 expression, and

(b) to detect the LFA-1 alpha subunit.

The invention also relates to a method of diagnosing the presence and location of inflammation resulting from the reaction of the specific defense system in mammals suspected of having inflammation consisting in

(a) incubating a human tissue sample with a composition containing a nucleic acid molecule capable of binding to a molecule selected from the group consisting of a LFA-1 alpha subunit DNA sequence; and a DNA sequence of the LFA-1 alpha subunit mRNA sequence of a gene for the LFA-1 alpha subunit, wherein the binding is capable of identifying a cell expressing * ICAM-1 or another natural ligand of LFA-1, and

(b) to detect the nucleic acid molecule.

The invention relates to a method for diagnosing the presence and location of inflammation resulting from the reaction of the specific defense system in a mammal suspected of having an inflammation consisting of:

(a) incubating a subject tissue sample with a composition containing a detectably labeled LFA-1 alpha subunit capable of identifying a cell capable of expressing ICAM-1 or another natural ligand of LFA-1, and

(b) to detect the LFA-1 alpha subunit.

The invention also relates to a method of diagnosing the presence and location of a tumor cell expressing ICAM-1 or another natural ligand of LFA-1 in a subject suspected of having a cell which consists therein :

(a) administer to the individual a composition containing a detectably labeled binding ligand capable of binding to ICAM-1 or the other natural ligand of LFA-1, the ligands being selected from the group consisting of: LFA -1-alpha subunit and functional derivative of the LFA-1 alpha subunit, and

(b) determine the binding ligand.

The invention also relates to a method of diagnosing the presence and location of a tumor cell expressing ICAM-1 or another natural ligand of LFA-1 in a subject suspected of having a cell which consists therein :

(a) incubate a subject tissue sample in the presence of a composition containing a detectably labeled binding ligand capable of binding to ICAM-1 or the other natural ligand of LFA-1, the ligands being selected from the group consisting of: LFA- 1-alpha subunit and functional derivative of the LFA-1 alpha subunit, and

(b) to detect the binding ligand bound to the ICAM-1 present in the tissue sample.

Ausführungsbelsplele

Fig. 1: shows a profile of reverse phase high pressure liquid chromatography separation (reverse phase HPLC separation) of LFA I alpha subunit triptin peptides. The elution was monitored by optical density at 280 nm (lower profile) and 214 nm (upper profile). The one-pointed peptides were subjected to protein microsequencing. The line across the profile indicates the percentage of acetonitrile.

Fig. 2: shows the restriction map of the LFA-1 alpha subunit CDNA clones. Restriction sites are BaI (B 1), Bam HI (B), Bgl I (Bg), CIa I (C), Eco RI (R), Hinc II (H), Nru I (N), Pst I (P) , Sen I (Sc) and Sma I (S). Arrows indicating the sequencing strategy are shown.

Fig. 3: shows the nucleotide and deduced amino acid sequence of the LFA-1 alpha subunit. The consequences of the triptin peptides and the transmembrane region are underlined by a strong or a shaded line. The putative serine phosphorylation sites are marked with a circle. The nucleotides in the З'-nontranslated region, which are underlined, correspond to an AIu 1 sequence.

Fig. 4: shows an alignment or alignment of the internal repeats of the LFA-1 alpha subunit. The consensus flanking sequence is shown over the ranked repeats, while the consonant divalent binding site and the binding sites described above are shown below. An asterisk indicates that more than one oxygen is involved in cation binding.

Fig. 5: shows an alignment of the human LFA-1 alpha subunit with other members of the integrin supergene family and the N-terminal of the murine LFA-1 alpha subunit. The LFA-1 and at least one integrin common residues are provided in a box. The section of the leukocyte insert is detectable. The boundaries of the homologous repeats containing the divalent cation binding sites are indicated by triangles. The protease site in the ECM receptor alpha subunits is indicated by arrows. The transmembrane area is underlined.

Figure 6: Shows a ranking and comparison of the L domain domain of the LFA-1 alpha subunit with homologous domains in von Willebrand factor (vWF), factor B, and CMP.

Fig. 7: shows a schematic representation of the evolutionary relationships of domains that are homologous to the L domain.

I. The nature of leukocyte adhesion proteins of the LFA-1 family

The three leukocyte adhesion proteins Mac-1, ρ 150.95 and LFA-1 differ in function and in expression on leukocyte subpopulations. Mac-1 and p150.95 are expressed on neutrophils and monocytes (Springer, TA et al., In "Biochemistry of Macrophages" (CIBASymposium 118); Pitman, London, pp. 102-126 [1986]) Differentiation of blood monocytes into tissue macrophages greatly increases expression of ρ 150.95 and decreases Mac-1 expression (Schwarting, R. et al., Blood 65: 974-983 [1985]; Hogg, N. et al., Eur Immunol. 16: 240-248 [1986]).

ρ 150.95 is also expressed on various types of activated T and B lymphocytes but is not expressed in blood on these cells (Kaligaris-Cappio, F. et al., Blood 66: 1035-1042 [1985]; Miller, LJ et al., J. Immunol, 137: 2891-2900 11986]; Keizer.GDua, J. Immunol 138: 3130-3136 [1987]).

Mac-1 and ρ 150.95 are expressed in an intracellular, vesicular compartment in the circulating neutrophils and monocytes, which are mobilized by inflammatory mediators to the cell surface (Todd, RF et al., J.CIIn. Invest. 74: 1280-1290 [1984 ); Springer, T.A. a "In: Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102 to 126 [1986]; Lanier, L.L. a. Eur. J. Immunol. 15: 713-718 [1985]; Yancey, K.B. a., J. Immunol. 135: 465-470 [1985]). This mobilization correlates with increased adhesiveness (Anderson, D.C., et al., Ann. Rev. Med. 38: 175-194 (1987)).

Monoclonal antibodies to Mac-1 or ρ 160.95 prevent neutrophil aggregation and adhesion to endothelial cells, protein-coated surfaces, bacteria, protozoan parasites and fungi (Harlan, JM et al., Blood 66: 167-178 (1985); Springer, TA et al. Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 [1986]; Dana, N. et al., J. Immunol 137: 3259 [1986]; Bullock, WS et al. J.Exper, 165: 195-210 (1987); Mosser, DM et al., J. Immunol., 135: 2785-2789 [1985]).

Mac-1 is also a receptor for complement component iC3b (Beller, DI et al., J. Exper. Med 156: 1000-1009 (19821). It has been demonstrated that detergent-soluble Mac-1 and ρ 150.95 are capable to bind to ІСЗЬ-Sepharose (Micklem, KH et al., Blochem.J.231: 233-236 [1985]).

LFA-1 is present on all leukocytes with the exception of a subset of macrophages. Monoclonal antibody blocking studies have shown that LFA-1 is important in T-lymphocyte mediated killing, T-helper lymphocyte responses, natural killing, and antibody-dependent killing (Springer, TA et al., Ann. Rev. Immunol. 5: 223-252 [1987 ]). Adhesion to the target cell is a step blocked by antibodies to LFA-1. Functional studies suggest that LFA-1 interacts with various ligands, one of which is ICAM-1 (Rothlein, R.u.a., J. Immunol.137: 1270-1274 [1986]).

It has been found that some cytotoxic T lymphocyte clones express similar levels of ρ 150.95 and LFA-1. Monoclonal antibodies to these LFA-1 and ρ 150.95 alpha subunits prevent killing by these CTL clones to a similar extent and are additive in their inhibitory activity (Keizer, GD et al., J. Immunol., 138: 3130-3136 [1987]). In addition, antibodies to ρ 150.95 alpha subunits have been shown to inhibit monocyte binding to endothelium (Keizer, G.D., et al., Europ. J. Immunol 17: 1317-1322 [1987]).

II. Cloning of the LFA-I alpha subunit

A variety of methods can be used to clone the LFA-1 alpha subgenus gene. One of these methods involves the analysis of a swing vector library of cDNA inserts (derived from an LFA-1 alpha subunit expressing cell) for the presence of an insert containing the LFA-1 alpha subunit gene. Such analysis can be performed by transfecting cells with the vector and then checking for expression of the LFA-1 alpha subunit. A preferred method for cloning the gene of the LFA-1 alpha subunit involves determining the amino acid sequence of an LFA-1 alpha subunit molecule or the triptin peptides of the molecule. For this purpose, LFA-1 alpha subunit molecules are preferably prepared by monoclonal antibody affinity chromatography of de . purified and purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis ("SDS-PAGE") and electro-olution (Miller, LJ et al., J. Immunol., 138: 2381-2382 [1987], a paper which is hereby incorporated by reference) The alpha subunit molecules are fragmented with cyanogen bromide or with proteases such as papain, chymotrypsin or trypsin (Oike, J. et al., J.BM. Chem. 257: 9751-9758 [19821; Liu, C., et al., Int. J. Pept Preferably, the alpha subunit is proteolytically digested with trypsin The resulting peptides are separated by reverse phase HPLC and subjected to amino acid sequencing, preferably by automated sequencers If it is possible to determine the complete amino acid sequence of the LFA-1 alpha subunit, preferably only the sequence of the peptide fragments of the molecule will be determined A preferred source of the LFA-1 alpha subunit is the Z elliptic SKW3. The sequence of amino acid residues in a peptide is referred to herein either using the commonly used three letter designations or by their single letter designation. A listing of these three-letter and one-letter designations is given in textbooks such as Biochemistry, Lehninger, A., OrIh Publishers, New York, NY (1970). If such a sequence is listed vertically, the amino terminal residue should be at the top of the set-up, while the carboxy-terminal residue of the peptide should be placed at the bottom of the set-up. Likewise, when positioned horizontally, the amino terminus should be on the left side, while the carboxy terminus should be on the right end.

The residues of amino acids in a peptide can be separated by hyphens. These thought or hyphens are only intended to facilitate the presentation of the sequence. As a purely illustrative example, the following amino acid sequence is given:

Gly-Ala-Ser-Phe

it indicates that an Ala residue is attached to the carboxy group of Gly, and that a Ser residue is linked to the carboxy group of the Ala residue and to the amino group of a Phe residue. The term further indicates that the amino acid sequence contains the tetrapeptide Gly-ALa-Ser-Phe. The term is not intended to limit the amino acid sequence to this one tetrapeptide, but is intended to include (1) the polypeptide having one or more amino acids attached to either its amino or carboxy terminus, (2) the tetrapeptide having one or more Amino acid residues bound to both its amino and carboxy terminus, (3) the tetrapeptide without additional amino acid residues. Once one or more suitable peptide fragments have been sequenced, the DNA sequences that they can code for are examined. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. (Watson, J.D., in: Molecular Biology of the Gene, 3rd ed., W.A. Penjamin, Inc., Menlo Park, CA [1977], pp. 356-357). Did peptide fragments are analyzed to identify sequences of amino acids that

are coded by oligonucleotides with the lowest level of degeneration. This is preferably done by identifying sequences containing amino acids encoded by only a single codon. Although occasionally an amino acid sequence can be encoded by only a single oligonucleotide, the amino acid sequence can often be encoded by any of a number of similar oligonucleotides. Importantly, although all members of this series contain oligonucleotides that can encode the peptide fragment and thus potentially contain the same oligonucleotide sequence as the gene that can encode the peptide fragment, only one member of the set contains the nucleotide sequence identical to the nucleotide sequence of the gene is. Since this member is present in the series or set and can hybridize to the DNA even in the presence of other members of the series, it is possible to use the unfractionated set of oligonucleotides in the same manner as a single oligonucleotide to clonidine the gene encodes the peptide.

A suitable oligonucleotide or series of oligonucleotides capable of encoding a fragment of the LFA-1 alpha subunit gene (or complementary to that oligonucleotide or array of oligonucleotides) is identified (using the method described above ), synthesized and hybridized by well-known methods, to a DNA or, preferably, a cDNA preparation derived from human cells capable of expressing the LFA-1 alpha subunit gene. Methods of nucleic acid hybridization are described by Maniatis, T. et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NYII982!) And by Haymes, B.D. a., (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC [1985]), which references are hereby incorporated by reference. The source of the DNA or cDNA used has preferably been enriched for LFA-1 alpha subunit sequences. This enrichment is most readily possible with cDNA obtained by extraction of RNA from cells producing high levels of the LFA-1 alpha subunit. Methods such as those described above or the like have succeeded in cloning genes for human aldehyde dehydrogenases (Hsu, LC et al., Proc. Natl. Acad. Sci. USA 82: 3771-3775 [1985]), fibronectin (Suzuki, S. et al , Eur.Mol.BIoI.Organ.J.4: 2519-2524 [1985]), the human estrogen receptor gene (Walter, P. et al., Proc. Natl. Acad. Sci. USA 82: 7889-7893 [1985]. ), tissue-type plasma activator (Pennica, D., et al., Nature 301: 214 to 221 [1983]), and human alkaline phosphatase placental complementary DNA (Kam, W. et al., Proc. Natl. Acad. Sci. USA 82: 8715-8719 [1985]).

Using the genetic code (Watson, JD, in Molecular Biology of the Gene, 3rd ed., WA Benjamin, Inc., Menlo Park, CA [1977]), one or more oligonucleotides can be identified each would be able to encode the tryptin peptides of the LFA-1 alpha subunit. The probability that a particular oligonucleotide will in fact represent the actual coding sequence of the actual LFA-1 alpha subunit may be determined by considering abnormal base-pairing relationships and the frequency with which a particular codon is actually used to encode a particular amino acid) eukaryotic cells. Such "codon usage rules" are described by Lathe, R., et al., J. Molec., BIoI. 183: 1-12 (1985) Using the "codon usage rules" of Lathe, a single oligonucleotide or a series of oligonucleotides carrying a theoretical " The oligonucleotide or set of oligonucleotides containing the theoretically "most probable" sequence containing the fragments of the L ^C A- or LJ-1 subunit will be identified as the most probable "nucleotide sequence capable of encoding the tryptin peptide sequences of the LFA-1 alpha subunit. 1 alpha-subunit, is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides capable of hybridizing to the "most likely" sequence or series of sequences An oligonucleotide containing such a complementary sequence , can be used as a probe to identify and isolate the gene of the LFA-1 alpha subunit (Maniatis , T. et al., Molecular Cloning. A Laboratory Manual [Molecular Cloning, A Laboratory Manual], Cold Spring Harbor Press, Cold Spring Harbor, NY [1982]).

In summary, therefore, it can be said that the actual identification of the peptide sequences of the LFA-1 АІрпь subunit allows the identification of a theoretical, "most probable" DNA sequence or series of such sequences capable of encoding such a peptide. By constructing an oligonucleotide that is complementary to this theoretical sequence (or by constructing a series of oligonucleotides that is complementary to the set of "most likely" oligonucleotides), one obtains a DNA molecule (or series of DNA molecules) can serve as a probe to identify and isolate the gene of the LFA-1 alpha subunit. Single-stranded oligonucleotide molecules complementary to the "most likely" tryptin peptide coding sequences of the LFA-1 alpha subunit have been synthesized using methods well known to those skilled in the art (Belagaja, R., et al., J. Bic! Chem. Manila, T. et al., Molecular Mechanisms in the Control of Gene Expression, Nierlich, DP et al., Hsg., Acad. Press, NY [1976]; Wu, R. et al., Proc. Nucl. Acid Res. Molec Biol. 21: 101-141 [1978]; Khorana, RG, Science 203: 614-625 [1979]). In addition, DNA synthesis can be accomplished by using automated synthesizers be performed.

It is possible to clone the gene of the LFA-1 alpha subunit from eukaryotic DNA preparations in which this gene is suspected. To identify and clone the gene encoding the protein of the LFA-1 alpha subunit, a DNA library or preferably a cDNA library is screened for its ability to hybridize to the oligonucleotide probes described above. Suitable DNA Präpa "Jte (such as human genomic DNA) is enzymatically cut or randomly sheared, and ligated into recombinant vectors The ^Fa:... Nkeit of these recombinant vectors to hybridize to the above-described oligonucleotide probes is then measured procedure for the hybridization for example, by Maniatis, T. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982), or Haymes, BT et al., Nucleic Acid Hybridization, A Practical Approach (Nucleic Acid Hybridization A Practical Method), IRL Press, Oxford, England (1985) The vectors capable of hybridization are then analyzed to determine the extent and nature of the LFA-1 alpha subunit sequences contained therein. Based on purely statistical considerations, ьіп gene, such as that encoding the molecule of the LFA-1 alpha subunit could be rt, can be clearly identified (via hybridization screening) when an oligonucleotide probe of only 18 nucleotides is used.

The LFA-1 alpha subunit monogenic gene obtained by the method described above can be operably linked to an expression vector and introduced into prokaryotic or eukaryotic cells to produce the protein of the LFA-1 alpha subunit , Techniques for these manipulations are described by Maniatis, T. et al. a., supra, and are well known.

III. Expression of the LFA-1 alpha subunit

The present invention is derived in part from the discovery of the cDNA sequence encoding the alpha subunit of the LFA-1 molecule. By operably binding this sequence (or a fragment of this sequence) to a functional promoter), it is possible to direct the expression of the alpha subunit of LFA-1 (or a functional derivative) in a cell or organism.

It is said that a nucleic acid molecule capable of "expressing" a polypeptide is "operably" containing nucleotide sequences containing transcriptional and translational regulatory information and operably linked to nucleotide sequences encoding the polypeptide " are. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence that is to become expression are linked so that expression of the gene is possible. The precise nature of the regulatory regions required for gene expression may vary from organism to organism, but generally must include a promoter region which, in prokaryotes, both the promoter (which directs the initiation of RNA transcription) and also contains the DNA sequences that signal the initiation of protein synthesis during transcription into the RNA. Regulatory regions in eukaryotic cells generally include a promoter region that is sufficient to direct the initiation of RNA synthesis.

It is said that two DNA sequences (such as the promoter region with its sequence and a sequence coding for the LFA-1 alpha subunit) are operably linked if the nature of the binding between the two DNA sequences is not (1) (2) the ability of the promoter region sequence to interfere with the transcription of the LFA-1 alpha subunit-encoding sequence or (3) affect the ability of the LFA-1 alpha subunit coding sequence by the sequence of the promoter region to be transcribed. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting the transcription of that DNA sequence. The present invention includes the expression of the LFA-1-Alpi.a subunit (or its functional derivative) in prokaryotic or eukaryotic cells. In order to express the LFA-1 alpha subunit (or its functional derivative) in a prokaryotic cell (such as E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to use the coding sequence of LFA-1. Alpha subunit operably linked to a functional prokaryotic promoter. These promoters may be either constitutive or preferably regulatable (ie, inducible or derepressible). Examples of constitutive promoters include the Int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol tocetyltransferase gene of pPR325, etc. Examples of inducible prokaryotic promoters include the right ones and left main promoters of bacteriophage λ (P _L and Pr), the E. coli trp, recA, lacZ, lad and gal promoters, the α-amylase (Ulmanen, I. et al., J. Bacteriol. 176-182 (1985)) and the B. subtilis 6-28 specific promoters (Gilman, MZ et al., Gene 32: 11-20 (1984)), Bacillus bacteriophage promoters (Gryczan, TJ, in: The Molecular Biology of the Bacilli], Academic Press, Inc., NY [1982]), and Streptomyces promoters (Ward, JM et al., MoI Gen Gen 203: 468-478, for a review of prokaryotic promoters Glick, BR (J.lnd.Microblol.1: 277-282 [1987]); Cenatiempo, Y. (Biochimie 68: 505-516 [1986]) and Gottesman, S. (Ann. Rev. G enet. 18: 415-442 [1984]).

Proper expression in a prokaryotic cell requires the presence of a ribosome binding site upstream of the gene coding sequence. Such ribosome binding sites are described for example by Gold, L. u. a., Ann. Rev. Microbiol. 35: 365-404 (1981).

If expression in a eukaryotic cell, e.g. Yeast, if desired, also in fungi, cells of mammals or plant cells, then a promoter capable of directing transcription in such a eukaryotic host must be employed. Preferred sukaryotic promoters include the mouse Metaalthionein I gene (Hamer, D. et al., J. Mol. Appl. Gen.1: 273-288 [1982]); the TK promoter of the Herpex virus (McKnight, S., Cell 31: 355-365 [1982]); SV40 prepromoter (Benoist, C., et al., Nature [London] 290: 304-310 [1981]; the yeast gal4 gene promoter (Johnston, SA et al., Proc. Natl. Acad. Sc. USA 79: 6971- 6975 [1982]; Silver, PA et al., Proc. Natl. Acad. Sc. USA 81: 5951-5955 [1984].) As is well known, translation of eukaryotic mRNA is initiated at the codon encoding the first methionine For this reason, it is advantageous to ensure that the binding between an eukaryotic promoter and a DNA sequence encoding the LFA-1 alpha subunit (or its functional derivative) does not contain intervening codons containing a methionine (ie AUG The presence of such codons results in either the formation of a fusion protein (when the AUG codon is in the same reading phase as the LFA-1 coding DNA sequence) or a phase shift mutation (if the AUG codon is not present in the same reading phase as the LFA-1 · coding sequence).

The DNA sequence encoding the LFA-1 protein (or its functional dorivate) when operably linked to a functional promoter is preferably prepared by a variety of suitable means, such as transformation, transfection, conjugation, protoplast fusion, electroporation, etc. introduced into a recipient cell.

The LFA-1 alpha subunit coding sequence and an operably linked promoter can be introduced into a recipient cell as a non-replicating DNA (or RNA) molecule that is either a linear molecule or, more preferably, a closed, covalent, circular molecule can. Since such molecules are incapable of autonomous replication, expression of the LFA-1 alpha subunit polypeptide may be by transient expression of the introduced sequence. Alternatively, permanent expression may be achieved by integration of the introduced sequence into the host chromosome.

Preferably, the introduced sequence is introduced into a plasmid or viral vector capable of autonomous replication in the host. For this purpose, a whole variety of vectors can be used. Among the factors of importance in the selection of a particular plasmid or viral vector are the ease with which the recipient cells containing the vector can be recognized and selected from the recipient cells that comprise the recipient

Vector not included; the number of copies of the vector desired in a particular host, and the question of whether it is desirable for the vector to be able to "sway" between host cells of different S. e., Among the preferred prokaryotic vectors are plasmids, such as those suitable for replication in E. coli (such as, for example, pBR 322, CoIE 1, pSC101, pACYC184, nVX) Such plasmids are described, for example, by Maniatis, T. et al., in: Molecular Cloning, A Leborytory Manual (Molecular Cloning Laboratory Manual), Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are described by Gryczan, T. (in: The Molecular Biology of the Bacilli! Molecular Biology of Bacilli), Academic Press, NY (1982), pp. 307-329. Suitable Straptomyces plasmids include pI101 (Kendall, KJ et al., J. Bacterlol. 169: 4177-4183 (1980) and Streptomyces bacteriophage such as "5C31 (Chater, KF et al., in: Sixth International Symposium on Actinomycetal Biology [6. International Symposium on Actinomycetidic Biology], Akademlai Kaido, Budapest, Hungary [1986], p.45-54). For a review of Pseudomonas plasmids, see John, J.F. a. (Rev. Infect. Dis 8: 693-704 (1986), as well as Izaki, K. (Jpn. J. Bacterlol. 33: 729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40.2 micron, etc. or their derivatives. These plasmids are well known in the art (Botstein, E., et al., Miami Wntr. Symp. 19: 265-274 [1982]; Broach, JR, in: The Molecular Biology of the Yeast Saccharomyces; Life Cycle and Inheritance. Saccharomyces: Life Cycle and Inheritance], Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p.445-470 [1981]; Broach, JR, Cell 28: 203-204 [1982]; Bollon, DP et al., J.CIIn. Hematol.Oncol. 10: 39-48 [1980]; Maniatis, T. in: Cell Biology: A Comprehensive Treatise, Vol.3 Gene Expression [Cell Biology: A Comprehensive Discussion, Vol.3 Gene Expression], Academic Press, NY , P.563-808 [1980]).

IV. Application of the LFA-1 alpha subunit or its fragments

The present invention provides the nucleic acid and protein sequences of the alpha subunit of the LFA-1 receptor molecule. This discovery allows the use of the recombinant DNA technique to produce the LFA-1 alpha subunit molecule. As will be explained below, one embodiment of the present invention relates to the use of the alpha subunit of the LFA-1 molecule as such, as an anti-inflammatory agent. In a preferred embodiment, the alpha subunit of the LFA-1 molecule is used in conjunction with its beta subunit. Such a compound or combination can be brought about by a variety of methods. For example, the beta-subunit of LFA-1 molecule can be produced separately from the LFA-1 alpha subunit and the two molecules then mixed together. However, it is more advantageous to produce the alpha and beta subunits of LFA-1 in the same host cell to facilitate their self-assembly to an LFA-1 heterodimer receptor molecule. The beta subunit of LFA-1 (which is common to LFA-1 and Mac-1) can be produced either by chemical synthesis or by recombinant DNA techniques (Kishimoto, TK, et al., Cell 48: 681-690 [1987 ]). The cloning of the beta subunit of LFA-1 is further disclosed in the related US Pat. Serial No. 019440, filed February 26, 1987, incorporated herein by reference.

One aspect of the present invention relates to the discovery of the nucleic acid and protein sequence of the alpha subunit of the LFA-1 receptor molecule. This discovery allows the use of the recombinant DNA technique to produce functional derivatives of the LFA-1 alpha subunit that can act as antagonists of cell adhesion. By "cell adhesion antagonist" it is meant any molecule capable of inhibiting the process of cell-line or cell-substrate adhesion. Whether a compound is an antagonist can be determined by a review of leukocyte aggregation Appropriate screening methods are described, for example, by Rothlien, R., et al., J. Exper Mod., 183: 1132-1149 (1986), which reference is hereby incorporated by reference, and antagonists of leukocyte aggregation can be used as anti-inflammatory agents.

By a "functional derivative" of the alpha subunit of LFA-1 is meant a compound having a biological (functional or structural) biological activity substantially similar to the biological activity of the alpha subunit of LFA-1 Examples of biological activity include the ability to bind to ICAM-1, another natural ligand of the LFA-1 molecule or the beta subunit of the LFA family of glycoproteins, such binding would inhibit adhesion-related events, such as lymphocyte binding to endothelial cells , antigen-presenting cells or target cells. It is said that one molecule is "substantially similar" to another molecule when both molecules have substantially similar structures or when both molecules have similar biological activity. The "functional derivatives" of the LFA-1 alpha subunit include both "fragments" and "variants" of the LFA-1 alpha subunit. The term "alpha subunit fragment of LFA-1" is understood to mean any one Polypeptide subset of this molecule. By the term "variant alpha subunit of LFA-1" is meant a molecule which is substantially similar in structure to either the whole molecule or a fragment thereof, provided that the "variant" has at least one biological activity activity of the alpha subunit of LFA-1 is either similar or inhibits LFA-1 activity. Thus, provided that a molecule possesses at least one biological activity that is either similar to or inhibits an activity of LFA-1, it is considered a "variant" of the alpha subunit of LFA-i, as that term is used herein even if one of the molecules contains one or more amino acids that are not present in the other, or if the sequences of the amino acid residues in the two molecules are not identical, for example, a verb Jung, containing one or more amino acid residues in each molecule would be the alpha subunit of LFA-1 found to be missing (or not containing) is considered to be a variant of the alpha subunit of LFA-1, if that compound had biological activity corresponding to a biological activity of the LFA-1 Alpha subunit of LFA-1 would be similar (or this subordinate). The term "biological activity" is intended to be "catalytic" like "Structural" activity, etc. (ie, the ability to bind to another molecule, such as ICAM-1, another natural ligand of the LFA-1 molecule, the beta subunit of LFA-1, or an anti-alpha subunit Bind LFA-1 antibody).

The present invention relates to a method for producing functional derivatives of the alpha subunit of the LFA-1 molecule. To obtain such derivatives, one needs only one DNA, RNA or (preferably) the cDNA sequence

muU) njsieran encoding the LFA-1 alpha subunit. Mutagenesis can be either random or site specific. In addition, mutagenesis may be induced either spontaneously or using chemical, radioactive or recombinant techniques.

The scope of the invention is also intended to include functional derivatives which lack certain amino acid residues or which contain altered amino acid residues as long as these derivatives have the ability to enhance or inhibit cellular adhesion.

Chemical mutagens include a base analog (such as 5-bromouracil or 2-aminopurine); Deaminating agents (such as nitrous acid, hydroxylamine, etc.); alkylating agents (such as methylmethanesulfonate, nitrosoguanidine, etc.) or intercolating agents (such as Acridincrange, Ethidium bromide, psoralen, etc.). The beam-induced mutation can be effected by such means as ultraviolet light, gamma rays, X-rays, etc. Methods for mutagenizing nucleic acid molecules are given by Miller, JH (in: Experiments In Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor , NY [1972]) and Silhavy, TJ et al (in: Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1984]).

Cellular specific mutagenesis can be used to produce specific mutations at desired sites of the nucleic acid encoding the LFA-1 alpha subunit. Briefly, such methods generally involve the synthesis of a synthetic oligonucleotide having a desired and defined DNA sequence. Methods for synthesizing such oligonucleotides are described by Itakura, K. u. a. (Ann., Rev. Biochem., 53: 323-356 [1984]). A nucleic acid molecule that does? LFA-1 alpha subunit protein or its functional derivative is generally subcloned onto a double-stranded vector such as M13, 0X174, etc., whose individual strands may be separated from each other. Then a single strand of the vector is incubated in the presence of the synthetic oligonucleotide. Since the DNA of the oligonucleotide is controllably defined, it is possible to construct an oligonucleotide capable of base pairing with any portion of the nucleic acid encoding the LFA-1 alpha-unsaturation unit. Once base pairing has occurred between the oligonucleotide and the single-stranded plasmid, one can expand the oligonucleotide using DNA polymerase to create a double-stranded DNA molecule which can then be capped by DNA ligase. When this double-stranded DNA molecule is introduced into a cell, semiconservative DNA replication results in the production of progeny molecules in which the DNA sequence of the oligonucleotide fragment has been included in the coding sequences for the LFA-1 alpha subunit. Alternatively, the LFA-1 alpha subunit of the present invention or its functional derivative can be prepared by chemical synthetic methods using the well-known Merriefield or other peptide synthesis techniques. Alternatively, such molecules can also be made by chemical synthesis of nucleic acid molecules (using, for example, the phosphodiester synthesis methods) which, after expression, result in their production.

For example, if one wishes to introduce a point mutation and an exogenous DNA sequence into a particular site of the LFA-1 coding sequence, or to make a deletion of nucleotides normally present in such a sequence, one would construct an oligonucleotide fragment carrying the mutation or sequence contains (or does not contain), and then apply the method described above. In order to introduce such a mutation or exogenous DNA sequence into a specific region of the LFA-1-alpha subunit-encoding nucleic acid, the mutation or the exogenous ΓιΊΑ sequence must be surrounded with flanking DNA sequences that are complementary to the DNA sequence of the Are areas whose mutagenesis is desired (Jenkins, F. et al., Bioessays 5: 244-247 (1986); Doerfler, W., Angew. Chern, Int. Ed. Engl. 23: 919-931 [1984]; Kaina, Biol.Zentralbl.99: 513-531 [1980]; Kunkel, Proc. Natl. Acad. Sc. USA 82: 488-492 [1985]; Nisbet, IT et al., GeneAnal.Techn.2: 23-29 [1985]; Hines, JC et al., Gene 11: 207-218 [1980]; Messing, J. et al., Nucl. AcldRes 9: 309 [1981]). Mutations can also be generated by using the recombinant DNA technique. For example, the nucleotide sequence of a nucleic acid molecule encoding the LFA-1 alpha subunit can be scanned to identify oligonucleotide sites recognizable by restriction endonucleases. Such endonucleases can then be used to specifically cut the nucleic acid sequence at a recognized site. By employing a restriction endonuclease which recognizes (and cleaves to) two positions in the LFA-1 coding sequence, it is possible to excise a fragment of the sequence encoding the LFA-1 alpha subunit. Alternatively, it is possible to use two different endonucleases for this purpose. By incubating the cleaved molecules in the presence of DNA ligase, it is possible to reseal the LFA-1 alpha subunit-encoding sequences and form a single sequence (which lacks the excised fragment). If there are no suitable recognition sites for the restriction nuclease in the LFA-1 alpha subunit coding sequences, such sites can be introduced into the sequences by the site-directed mutagenesis method described above. Alternatively, mutations can be introduced by cleaving the sequence encoding the LFA-1 alpha subunit and "nibbling" the free termini with an exonuclease, by such treatment it is possible to have not only deletions but also phase shifts and other types of It is also possible with this technique to introduce novel restriction endonuclease sites into the sequence encoding the LFA-1 alpha subunit Methods for the application of restriction endonucleases, DNA ligases and fxxonucleases are described, for example, by Maniatis, T. et al. Molecular Cloning, A Laboratory Manual [Molecular Cloning, Laboratory Manual], told Spring Harbor Laboratory, Cold Spring Harbor, NY [1982]).

Recombinant DNA methods can also be used to prepare fusion proteins composed of the LFA-1 alpha subunit protein (or its functional derivative) and a novel polypeptide. This novel polypeptide is not limited to a particular polypeptide and may have either a single amino acid or a set or a permutation of amino acids. Such a fusion molecule may be prepared by ligating a DNA sequence encoding the novel polypeptide to a DNA sequence encoding the LFA-1 alpha mammalian entity (or its functional derivative), in a manner such that no phase shift mutation is introduced. Examples of preferred polypeptides that can be fused to the LFA-1 alpha subunit gene (or its functional derivative) include eukaryotic or prokaryotic signal sequences (Gilbert, W., et al., U.S. Patent No. 4,411,994; Casadaban, M., et al., Proc Natl. Acad. Sci USA 76: 4530-4533 [1979]) or polypeptides which confer the stability, biological

Extend or decrease the half-life or potency of the LFA-1 alpha subunit (or its functional derivative). An excellent review of the methodology of gene fusions is given by Silhavy, T.J., et al. (in: Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1984]). In response to immunization with fragments of the LFA-1 alpha subunit, antibodies (especially monoclonal antibodies) can be elicited. These antibodies can be used to prevent the binding of certain leukocytes to endothelial cells, antigen-presenting cells or target cells, and thus can be used as anti-inflammatory agents.

Using the methods described above, fragments of the LFA-1 alpha subunit can be prepared and tested to see if they are antagonists of cell adhesion. Fragments found to be antagonists of cell adhesion can be used as anti-inflammatory agents in accordance with the present invention.

The present invention derives in part from the discovery that leukocyte-substrate adhesion and leukocyte-endothelial cell adhesion result from interactions involving the LFA-1 receptor. Since cellular adhesion is necessary for leukocytes to migrate to sites of initiation and / or to exert various effector functions that contribute to inflammation, agents that inhibit this cellular adhesion attenuate or prevent inflammation. The LFA-1 receptor molecule is present on the surface of leukocyte cells. The adhesion of these cells to monolayers of endothelial cells is partially mediated by LFA-1.

The interaction of LFA-1 molecules with their natural ligands (such as ICAM-1, etc.) is central to cellular adhesion. Through the process of adhesion, lymphocytes are able to continuously monitor an animal for the presence of foreign antigens. Although these processes are usually desirable, they are also the cause of rejection of organ transplants, rejection of tissue grafting, and many autoimmune diseases. Thus, any means to attenuate or inhibit cellular adhesion would be highly desirable in organ transplant recipients, tissue grafts, or patients with autoimmune diseases. Agents that counteract these interactions are very useful as anti-inflammatory agents in mammals. Significantly, these agents differ from the general antiinflammatory agents in that they can selectively inhibit adhesion and have no other side effects such as nephrotoxicity present with conventional agents. Agents that are capable of binding to the alpha subunit of LFA-1 or to ICAM-1 or to another natural ligand of the LFA-1 molecule can thus be used to reduce organ or tissue rejection Transplant host disease (ie, to prevent host tissue rejection caused by engraftment of bone marrow or other lymphocyte-containing or tissue-producing tissue) or to modify autoimmune responses by affecting cell-bound LFA-1 without the risk of side effects (such as the interaction with the cell-bound ligands, etc.). Importantly, the use of agents capable of recognizing such molecules enables organ transplants to be performed even between patients with HLA mismatch.

Agents that interfere with the ability of the LFA-1 receptor molecule to bind to the natural binding ligands may thus interfere with all of the obstructed LFA-1-dependent functions. Thus, these agents can serve as anti-inflammatory agents of the present invention. These agents include the LFA-1 alpha subunit, LFA-1 (alpha and beta subunits), and antibodies capable of binding to the LFA-1 alpha subunit or fragments of this subunit. All of these means can be used in accordance with the present invention. The anti-inflammatory agents of the present invention are useful in the treatment of inflammation caused by the reaction of the specific defense system. The term "specific defense system" is understood to mean the component of the immune system that responds to the presence of specific antigens, such as lymphocytes and macrophages, which is said to result from a reaction of the specific defense system when the inflammation occurs caused, mediated, or associated with a specific defense system response Examples of inflammation resulting from a specific defense system response include the response to antigens such as rubella virus, autoimmune diseases, T-cell mediated, delayed hypersensitivity (such as visible, for example, to those who are "positive" in the mantaux test), organ or tissue rejection, host-plant transplantation disease (ie, host tissue rejection caused by transplantation of bone marrow or other lymphocyte-containing or tissue-producing tissue), etc. The ability of the LFA -1 alpha subunit and its functional derivatives to counteract these inflammatory responses provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythemotosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms diabetes, Reynaud syndrome, rheumatoid arthritis, etc.

Since LFA-1 is expressed on cells capable of binding to endothelial tissue, administration of the LFA-1 alpha subunit or LFA-1 (alpha and beta subunits) to a patient presents a measure of resolution In addition, the method provides diagnostic information on the amount and distribution of the binding ligands of the LFA-1 receptor molecule present on the visualized tissue, in which application the LFA-1 alpha subunits become (or LFA-1 alpha-beta receptor molecules) by the use of radioisotopes, affinity tags (such as biotin, avidin, etc.), fluorescent labels, paramagnetic atoms, etc. The antibodies (or fragments thereof) can be detected by the use of radioisotopes , Enzyme labels, fluorescent labels, paramagnetic labels, electron density labels, toxin labels, etc. are detectably labeled Preferred toxin labels include diphtheria toxin, ricin and cholera toxins. The administration of these labeled molecules to a person identifies the sites of inflammation. Such detectable labels can also be used to check the status of a patient's immune system. A review of the use of antibodies in diagnostic visualization is given by Grossman, H. В., Urol. Clin. North Amer. 13: 465-474 (1986), Unger, E. C, et al. a "Invest. Rddlol. 20: 693-700 (1985) and Khaw, B.A., u. a., Science 209: 295-297 (1980).

The ability of leukocytes to spontaneously migrate to sites of inflammation is dependent on LFAO (Keizer, GD, et al., Eur.J. Immunol 17: 1317-1322 (1987)). This migration can be achieved by administration of LFA-I-alpha It has also been found that the ability of leukocytes to adhere to endothelial cells is dependent on LFAO Each of the anti-inflammatory agents of the present invention may be used to inhibit LFA-1 (alpha and beta subunit) subunits Suppression of these activities are used.

ICAMs (such as ICAM-1) are recognized by certain human viruses (particularly rhinoviruses of the major type) that bind to ICAM-1. These viruses bind to human cells due to this recognition and thus transmit the virus infection. A key step in the etiology of viral diseases is therefore the interaction between these cellular receptors and the virus.

Agents which suppress, compete with, or inhibit the ability of a virus to bind to an ICAM molecule are therefore of use in the treatment of viral (especially rhinovirus) infections. One aspect of the present invention therefore relates to the ability of the alpha subunit of LFA-1 and its functional derivatives to interact with ICAM-1 and thereby prevent either cell-virus binding and viral infection or the severity or duration of a virus to attenuate or shorten such infection.

Of particular interest to the present invention are functional derivatives of the alpha subunit of LFA-1, such as the solubilized forms of the alpha subunit of LFA-1, fragments of the alpha subunit of LFA-1, etc. These agents are given to a recipient patient preferably as a heterodimer containing the molecule in association with a molecule of the beta subunit of the CD-18 family. The above-described goal of treating a viral infection can be achieved with a single agent or with a combination of more than one agent.

For the purpose of treating viral infections, the agent (s) of the present invention are (are) to be administered to a recipient patient (for example, by intranasal agents) in a dosage sufficient for the agent (s) to suppress the ability of a virus these can compete or prevent them from binding to an ICAM molecule. This dosage will generally be from 0.01 pg / kg up to 1 mg / kg body weight of the patient (for each individual agent), although larger or smaller amounts may be used.

For the purpose of treating a viral infection, administration of this agent (s) may be either "prophylactic" or "therapeutic." In prophylactic administration, the agent (s) will be given before the time of infection (п., before, at or shortly after infection), but before the onset of symptoms of viral infection. The prophylactic administration of the agent (s) serves to prevent or alleviate a subsequent infection. In therapeutic administration, the agent (s) will be at the onset of a symptom of the actual viral infection (or a short time thereafter) (such as the occurrence of a virally induced nasal congestion, etc. or in detection of the virus in body fluid or antibody detection against the virus are directed, in the serum of the infected patient, etc.). Therapeutic administration of the agent (agent) serves to attenuate any actual infection and thereby reduce its severity or duration.

V. Administration of the LFA-1 alpha subunit

The therapeutic effects of the LFA-1 alpha subunit can be achieved when a patient has the LFA-1 receptor molecule (alpha and beta subunit). The entire molecule of the LFA-1 alpha subunit or its therapeutically active, functional derivatives are administered. These molecules can be obtained either synthetically or by use of the recombinant DNA technique or from natural sources. Fragments of the LFA-1 receptor or its alpha subunit can also be obtained by proteolysis. The therapeutic benefits of these molecules can be enhanced by the use of functional derivatives having additional amino acid residues added to enhance coupling to the carrier or to enhance activity.

The molecules of the present invention are referred to as being "substantially free of natural contaminants" when preparations containing them are substantially free of substances with which these products normally and naturally occur.

In treating a patient with the therapeutic molecules of the present invention, the dosage of the agent administered will vary widely depending on such factors as age, weight, height, sex, general medical condition, previous medical treatment, etc. of the patient. In general, it is desirable to administer to the recipient a dosage of the LFA-1 alpha subunit (or its functional drug) that ranges from about 1 pg / kg to 10 mg / kg (body weight of the patient), although also lower or higher dosages can be administered.

The molecules of the present invention can be administered to the patient intravenously, intramuscularly, subcutaneously, enterally or parenterally. The administration may be a continuous infusion or by single or multiple boluses.

It is envisaged to administer the anti-inflammatory agents of the invention to the recipient persons in an amount sufficient to suppress the inflammation. An amount is said to be sufficient to suppress inflammation if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent the inflammation. The anti-inflammatory agents of the present invention may be administered either prior to the onset of inflammation (so as to suppress the expected inflammation) or after the onset of inflammation. A composition is said to be "pharmacologically acceptable" if its administration by the recipient patient can be tolerated Such agent is administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. The molecules of the present invention may be used in accordance with known methods to prepare pharmaceutically acceptable compositions combining these substances or their functional derivatives in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, including other human proteins, e.g. Human serum albumin are described, for example, in Remington's Pharmaceutical Sciences (16th Edition), Osol, A., Ed., Mack, Easton, PA (1980). In order to produce a pharmaceutically active composition suitable for a

effective administration, these compositions contain a therapeutically effective amount of the LFA-1 alpha subunit or its fragments or functional derivatives together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of the effect, controlled release preparations may be obtained by the use of polymers which complex with or complex with the LFA-1 alpha subunit or its fragments or functional derivatives absorb. Controlled delivery may be by selection of suitable macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate) and the concentration of macromolecules, as well as the methods of inclusion so as to control release. Another possible method of controlling the duration of action of controlled release preparations is to include molecules of the LFA-1 alpha subunit, fragments or functional derivatives thereof in particles of a polymeric material such as polyesters, polyamino acids, hydrogels, Poly (lactic acid) or Ethylenvinylazetatkopolymere. As an alternative to incorporating these agents, it is possible to include these molecules in microcapsules prepared, for example, by coacervation or by interfacial polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly (methyl methacrylate) microcapsules, or in colloidal drug delivery systems, for example liposomes, albumin microspheres , Microemulsions, nanoparticles and nanocapsules in macroemulsions. These techniques are described in Remington's Pharmaceutical Sciences (1980). Having generally described this invention, it is now to be more readily understood by reference to the following examples, which are presented for purposes of illustration and the present invention unless otherwise specified to restrict in any way.

example 1 Purification of the LFA-1 alpha subunit

LFA-1 is expressed on the surfaces of SKW3 leukocyte expression. The LFA-1 alpha subunit molecule was purified from SKW-cells by monoclonal antibody affinity chromatography according to the method of Miller, LJ, et al., J. Immunol., 137: 2891-2900 [19871] (this reference is incorporated herein by reference ). The monoclonal antibody, TS1 / 22, directed against the alpha subunit of LFA-1 was purified and coupled (using cyanogen bromide) to CL-4 B-Sepharose (Pharmacia) with 2 mg MAB per ml packed bed. SKW3 cells (42.2 g) obtained from the MIT culture collection were dissolved in 300 ml of lysis buffer (Kurzinger, K., et al., J. Biol. Chem. 217.12412 to 12418 [1982]), and the lysate was then co-cultured 1600Ox g rotated for a period of 2 hours. Subsequently, the supernatent layer was successively passed through a precolumn of activated and quenched CL-4B Sepharose and through a TS 1/22 Sepharose column. The TS1 / 22 column was washed sequentially (Kurzinger, K., et al., J. Biol. Chem. 257: 12412-12418 [1982]), and the LFA-1 alpha subunit was treated with 50 mM triethylamine, 0.5M NaCl, 0.1% Triton X-100.1 mM iodoacetamide, 10 units / ml protein and 0.025% NaN ₃ , pH 11.5, and the pH was immediately neutralized. The fractions containing the LFA-1 alpha subunit were pooled, lyophilized and precipitated in 5 volumes of ethanol at -2O ⁰ C overnight. Purified protein was reduced and alkylated (Law, SK Α, et al., EMBO J. β: 915-919 [1987]) and subjected to preparative SDS-polyacrylamide gel electrophoresis. The band corresponding to the LFA-1 alpha subunit was visualized with 1M KCl, excised and electroeluted (foliegrino, MA, et al., CIIm Immunol Immuno., 3: 324-333 [1975]). The purified alpha subunit was lyophilized and precipitated in 4 volumes of ethanol at -20 ° C overnight. The pellet was resuspended and digested with 1% trypsin (Wong, WW, et al., Proc Natl Acad ScI, USA 82: 7711-7715 [1985]). The tryptin fragments were then isolated by HPLC (high performance liquid chromatography) (Beckman) on reverse phase column C4 (Vydac). The peptides were eluted on an acetonitrile gradient of 0 to 60% in 1% trifluoroacetic acid. Several peaks were isocratically rechromatographed in an acetonitrile concentration determined by the following equation:

F = (0.9E) -2

where F is the volume percent of acetone Url under isocratic conditions for a peptide that eluted during the linear gradient at E percent (Lathe, R, et al., J. Molec. Biol. 183: 1-12 [198S]). Tips were collected in 1.5 ml polypropylene tubes and concentrated to less than 50 μΙ. Eight tips were microsequenced. The sequence of a peptide, L64, was used to construct a single oligonucleotide according to the suggestions of Lathe, R., et al. (J. Molec.Blo.183: 1-12 [1985]):

S 'GGGATGTTGTGGTCATGGATGGTGGGCTCAAT-S'

In summary, LFA-1 was first isolated from SKW3, a T-lymphoma cell strain, lysate by monoclonal antibody affinity chromatography using an antibody directed against the alpha subunit. SDS-PAGE fractions had the alpha and beta subunits. The alpha unit was further purified by preparative SDS-PAGE and the purified alpha subunit was digested with trypsin and the peptides were isolated by reverse phase HPLC. The peptide sequence of nine peaks was determined by microsequencing (Figure 1). The peptide sequence having the lowest codon redundancy was used to specify a single oligonucleotide sequence that utilized the most common human codons (Latho, R., et al., J. Molec BIoI 183: 1-12 [1985]). The consequences of the tryptin peptides of the LFA-1 alpha subunit are shown in Table I.

Table I Consequences of Tryptlnpeptlden the LFA-1-Alpha-Unterelnhelt

Residues amino acid sequence

95-107 XXDQ (N) TYLSGL (E) YLF

199-210 HMLLLTNTFGAI

254-260 YIIGIGK

405-413 VLLFOEXQG

494-503 GEAITALTXI

541-554 IEGTQVLSGIQXFG

564-576 X (L) E (G) D (V / G) LADVAVGAE

803-817 KVEMLKPHSEEIXVS (T)

929-946 I (E / Q) PSIHNIP XLEAVX ''.

Brackets indicate ambiguities in the sequence. The underlined residues were used to generate an oligonucleotide probe.

Example 2 cDNA Cloning

Recombinants (5x10 ⁵ ) from an XgMO library selected for cDNA inserts greater than 2kb and produced from PMA-induced HL-60 cells (PMA-phorbol myristic acid) (Corbi, A., et al., EMBO J. 6 : 4023-4028 [1987]) were plated at a density of 50,000 recombinants per 150 mm plate. The library was amplified (Woo, SLC, Mot. Enzymol. 69: 389-395 [1979J, and nitrocellulose filters were processed according to the guidelines of Benton and Davis (Benton, WD, et al., Science 196: 180-182 Ц977]). the filters were prehybridized in 6x SSC (0.6M NaCl, 0,06MNatriumzitrat), 0.05% of potassium phosphate and Natriumpvrophosphat, 0, e% SDS, 6 x Denhardt's reagent and 100 pg / ml salmon sperm DNA at 42 ⁰ C overnight. the single oligonucleotide (sequence shown above) was labeled with P-ATP V- ³² using polynucleotide kinase. the filters were hybridized overnight in a Vorhybridisationspuffer and then washed sequentially in 6 x SSC, 0.05% Natriumpyrophospnat for 15 min at 5O ⁰ C Filters were exposed to preexposed XAR-5 fumes for between 6 and 20 hours, phages which gave signals on duplicate filters were plaque purified, and their cDKV inserts were electrophoresed on agarose gels.

The 32mer oligonucleotide probe was used to isolate twenty clones from a size-selected \ gt10 cDNA library constructed from PMA-stimulated myeloid cells (Corbi, A., et al., EMBO J. 6: 4023-4028 [1987]). , It has already been shown that these cells synthesize the LFA-1 alpha subunit (Miller, LJ, et al., J. Immunol 139: 842-847 [1987]). Insert size was determined and the longest clone, X5L5, was restriction-mapped (Figure 2). This clone contained the nucleotide sequence corresponding to the oligonucleotide probe (85% identity to the "best guess" probe.) The nucleotide probe also encoded the tryptin peptide that was determined by microsequencing (16 of 16 residues) that included and went beyond the oligonucleotide However, this clone did not encode the complete protein because not all of the tryptine peptide sequences were present in open reading frames The SLI S'-I.Okb-EcoRI fragment was used to rescreen additional 5x10 ⁶ recombinates, an additional 14 clones were identified, and the clone A3R1 had an identical restriction map in the overlapping regions and additionally contained a 1.0 kb 5 'fragment The nucleotide sequence of this additional fragment was determined in Fig. 2).

Example 3 Restriction Kartlerung and Nukleotldsequenzlerung

Restriction maps of selected clones were determined by duplicate and partial digestions (Maniatis, T., et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1982]). Restriction fragments were sulbcloned into either M13mp18 or M13mp19 (Messing, J., Met. Enzymol., 101: 20-78 [1983]) or pGEM-3Z, -4Z or -7Z (Promega). Deletions of the fragments in pGEM are made using exonuclease III or S1 (Henikoff, S. Gene, 28: 351-359 [1984]). Sequencing was done by the dideoxy termination method (Sunger F., et al., Proc. Natl. Acad. Sc. USA 74: 5463-5467 [1977]). The coding sequence of the 5 '' up I З 'non-translated regions were determined to be 100%, 83.1% and 33.7%, respectively, in both orientations.

The overlapping clones of cDNA contain 5139 nucleotides (Figure 3). There is an open reading key of 3510 nucleotides, a 5'-nontranslated region of 94 nucleotides, and a З'-nontranslated region of 1535 nucleotides containing a polyadylation site 15 nucleotides in front of the poly (A +! Tail within the З'-untranslated region There is a typical AIu 1 repeat consisting of two tandem-related sequences, each terminated by a Α-rich segment (Hardman, N., Blochem J.234: 1-11 [1986]). Sequence is 304 nucleotides Itng and is 78.8 kg identical to the normal sequence of the Alu family.

Example 4 Southern and Northern transfer

Southern transfer was performed as described by Corbi, A., et al. (J. Exper. Med. 167: 1597-1607 [1988]). On a 1.0% formaldehyde gel, 20 cc per ml of total cellular RNA isolated from either SKW3, U937, B4 or EJ cells were electrophoresed and transferred to nitrocellulose (Current Protocols in Molecular Biology ( Laufenda Molecular Biology Protocols), Green Publishing Associates and Wiley-Inte'.oience, New York). The nylon membrane (Zeta probe [Biorad]) was prehybridized and hybridized in 2x SSC, 1 x Dehnhardt's reagent solution, 0.1 % SDS and 10 μg / ml Herring sperm DNA. A 1.8kb EnoRi probe from the 5 'end of the cDNA clone, A3R1, was labeled by single strand break-through transatlation and used as a probe.

Northern transfer analysis revealed a message of 5.5kb in SKW3, U937, and IB4 cells, but no signal was detected in EJ cells. The cell distributions agree well with the size of the cDNA clone and the cell surface expression of LFA-1. The Southam transfer analysis using the 1.2kb 5 'EcoRI-BamHI fragment from clone X2L2 hybridized to two fragments of 10 and 8kb (Corbi, A., et al., J. Exper. Med. 167: 1597 until 1607 [1988]). A genomic clone isolated from a cosmid library has two fragments of identical length. These fragments are contiguous, demonstrating that LFA-1 is a single copy gene.

Example 5 Computer Analysis

For homology searches and alignment of sequences, the Microgenie DNA program (Beckman), FASTP (Wilbur and Sigman) on the NBRF and NEW databases (National Biomedical Research Foundation, Washington, DC) and FASTP with the SWISS PROT database (Bionet) used. These alignments were then optimized using ALIGN (NBRF) (5585) and GENALIGH (Bionet).

Hydrophobicity was determined using the Microgenie DNA program as well as PER-PLOT (5792) (University of Wisconsin Genetics Computer Group). The hydrophobicity analysis of the amino acid sequence revealed that the LFA-1 alpha subunit is a typical transmembrane protein having a hydrophobicity signal sequence, an extracellular region, a single hydrophobic transmembrane region, and a short cytoplasmic tail. The N-terminal residue of human LFA-1 was identified by homology to the N-terminal of murine LFA-1 (Figure 5). Human LFA-1 has an identity of 55% with mouse LFA-1 over the first 20 amino acids. A classical signal peptide with a conserved sequence (Ala-X-Ser / Pro) for the cleavage peptidase precedes the N-terminal sequence. There are three putative upstream transcription initiation sites (ATG) in the raster. Generally, use of the first initiation site at nucleotide position 89 (Kozak, M., Nucl. Acid Res. 12: 857-872 [1984]) is preferred, yielding a twenty-five residue signal sequence with multiple polar groups in the vicinity of N-terminal residues, as is typically found in signal sequences (von Heijne, G., J. Molec., Vol. 173: 243-251 [1984]). All 117 amino acids determined by microsequencing of tryptine peptides were found in the transferred open reading frame, confirming the authenticity of the cDNA clones. Taken together, these results demonstrate that the LFA-1 alpha subunit has an N-terminal 29 + amino acid signal sequence, an extracellular domain of 1063 residues, a transmembrane domain of 29 amino acids and a cytoplasmic C-terminal tail of 53 amino acids.

Seven repeats of 49-63 amino acids are within the extracellular domain. The degree of internal identity and statistical significance among these internal repeats between the three last repetition greatest, 24-33% for ρ <10 ~ ² to <10 "*. The central area of these latter three repeats has homology with several divalent cation binding sites (Figure 4) Since previous studies have shown that Mg ^{2+ is} continuous or at lower concentration in conjunction with Ca ²⁺ necessary for the functional integrity of the protein (Rothlein, R., et al., J. Exper. Med : 1132-1149 [1986]), the homology of the three tandem repeat divalent cation-binding templations suggests that LFA-1 binds divalent cations.The repeats HV do not have the divalent consensus cation-binding site, but have flanking regions obtained.These structural similarities open the possibility that these replications have resulted from a Dup'ikationsereignis.The mature protein has Mr = 1 26,193, and twelve N-linked glycosylation sites (Asn-X-Thr / Ser) are in the extracellular domain. Previous studies have shown that at least five of these cells are glycosylated and have oligosaccharides with M, = 5000 to 10,000 daltons, which is typical of the complex, N-linked carbohydrate (Dahms, NM, et al., J. Immunol., 134: 3978-3986 (1985)). Thus, the use of 5 to 10 of these glycosylation sites would predict a molecular weight consistent with the molecular weight determined by SDS-PAGE.

The determination of the N-terminal of mouse LFA-1 (Girma, J.-P., et al., Blood 70: 605-611 [1987]) and the primary structure of the common beta-subunit of LFA-1 / Mаs-1 / р 150.95 has suggested that the LFA-1 alpha-hypnosis is a member of the integrin family. The LFA-1 alpha subunit has a surprising homology (35% identity) to the alpha subunits of ρ 150.95 and Mac-1 and to a lesser extent to the alpha subunits of the ECM receptors (25% identity). On the other hand, FNR, VNR and Hb are 40% identical to each other. In addition, the leukocyte integrins also contain an insert of approximately 200 amino acids near the N-terminal region of the protein, which is absent in the three sequenced ECM receptors (Figure 5). In addition, the region in which VNR, FNR and gpllb / IIIa have a protease site is absent, both at LFA-1 and alpha subunits of ρ 150.95 and Mac-1, and correlates with the absence of alpha proteolytic processing Subunits of the LFA-1 family. Overall, these structural features defined two subfamilies of alpha subunit integrins, leukocyte integrins and ECM integrins. All Integnn family alpha subunits, like LFA-1, have similar repeats containing putative divalent cation binding sites (Corbi, A., et al., EMBO J. 6: 4023-4028 [1987]; Poncz, M., et al., J. Blol.Chem.2e2: 8476-8482 [Ti987]; Suzuki, S., et al., Proc. Natl. Acad. Sci. USA 83: 8614-8618 [1986]; Argaves, WS, et al., J. Cell BIoI : 1183- '· 1190 [1987], Ruoslatni, E., et al., Science 238: 4.91-497 [1987]). However, only three of the repeats in LFA-1 and ρ 150 contain putative metal binding sites, while in the ECM receptors all four repeats contain metal binding sites. The putative divalent cation binding sites of LFA-1 and the other integrins are related to but not identical to the previously described divalent cation binding sites of the integral membrane proteins. There are several types of divalent cation binding sites. The first set of such sites has an alternating DXD (NbX-D (N) structure, and these sites are found in troponin C, parvalbumin (5256), galactose binding protein, etc. These sites form an EF loop structure and have 6-7 chelating sites. The putative binding sites on LFA-1 and the other integrins also have a DXD (N) -XD (N) primary structure, with a series of G residues between the chelating residues, but the remainder in the Z position is passed through In addition, the alpha helix in front of and behind the calcium binding site seems to be absent in the integrin sites (Corbi, A., et al., EMBO J. 6: 4023-4028 [1987]) The significance of this change is unknown but partly be the cause of the preference of these sites for Mg ²⁺ .

A search of the NBRF and Swiss protein databases revealed that this LFA-1 family specific region, termed the "L" domain, had significant homology with the Al domain of human vWF, human complement factor B and These similarities extend the full length of the L domain. Since vWF has three A domain repeats (A1, A2, A3) (5790), the domain is in the complement factor of similar domain in the complement component 2 (C2) (5789). analogous, and CMP has 2 repeats (5778), comparing the LFA-1 L domain to all of these domains using the ALIGN program, with the exception of C2, all of which were statistically significant (Figure 6).

These similarities imply a possible functional domain for LFA-1. First, the A1 domain of vWF binds to glycoprotein I b and heparin (5646), while domains A1 and A3 are both involved in binding to collagen. Second, the homologous domain is located in Factor B on the C-terminal side of the cleavage site where Factor B is cut to Factor Bb, and on the N-terminal side of the Serine Protease (Mole, JE, et al., J.Biol.Chem.259: 3407-3412 (1984); Bently, DR, Blechern J. 239: 339-345 [1986]). This domain is therefore in the region expected to interact with the ligand, C3b. The structure of the collagen matrix protein has been partially determined and has two repeats separated by a sequence similar to the epidermal growth factor (Argaves, W.S., et al., Proc. Natl. Acad. Sci. USA 84: 464-468 [1987]). CMP interacts with collagen (Argaves, WS, et al., Proc. Natl. Acad. Sc. USA 84: 464-468 [1987]) as well as with cartilagin proteoglycan (Fig. 7) (Paulsoon, M., et al., Collagen Rel.Res.4: 219-229 [1984]).

LFA-1, Mac-1 and ρ 150.95 were recently localized to chromosome 16p11 (Corbi, A., et al., J. Eхрѳг., Med. 167: 1597-1607 (1988).) Structural similarity and proximity of genes , which encode the leukocyte-integrin alpha subunits, suggest that a primordial gene duplicated and resulted in the formation of at least two branches of the integrin alpha subunits, the leukocyte integrins and the ECM integrins into the primordial gene of the leukocyte integrin Alpha subunit, a domain of 180 amino acids was inserted, and then the gene duplicated and resulted in the formation of LFA-1, Mac- ¹ , and ρ 150.95 The similarity of these domains to the L domain of LFA-1 In addition, these homologies imply that a primordial domain has been inserted into more ⁰ '' * ^p . •. One, and then different recognition factors in hemostasis, complement activation development and immune response.

These structural differences between the two groups of alpha subunit integrins correlate with the difference in the recognition site of the functional domains, as RGD-containing peptides block the binding of FNR and gpllb / IIIa to the corresponding ligands (Ruoslathi, E., et al., Science 238: 491-497 (1987)), while an RGDS peptide does not inhibit the interaction of LFA-1 with ICAM-1. In contrast to the ligands of the ECM integrins, ICAM-1 itself has no RGD peptide sequence and is a member of the immunoglobulin superfamily. Both structural and functional data suggest that the integrin superfamily can be subdivided into two subfamilies of alpha subunits. However, these β ₂ -substituted structural and functional properties of the alpha subunits need not be unique to the subfamily, as various other integrin alpha subunits may also have structural properties similar to those of the LFA-1 family, such as the Absence of a proteolytic cutting site (Charo, IF, et al., Proc. Natl Acad. Sc. USA 83: 8351-8355 [1986]). The elucidation of the structure of the LFA-1 alpha subunit revealed novel developmental relationships between the integrin alpha subunits, suggesting a possible functional domain, and demonstrated that the LFA-1 alpha subunit is integrin Belongs to superfamily but contains an additional domain. This L domain and homologous domains represent a protein "domain" family that is of functional importance Since the recognition site of LFA-1 is not RGD, the L domain may be of functional significance in the LFA-1 alpha Subunit and the other leucocyte integrins, Mac-1 and ρ 150.95, however, since LFA-1 participates in a large number of leukocyte functions and has more than one ligand, (Rothlein, R., et al. Immunol 137. ", 270-1274 (1986)), it is possible that more than one functional domain exists in the LFA-1 alpha subunit. It is also interesting to analyze the interactions that the cytoplasmic tail engages with the cytoskeleton, and what role this interaction plays in signaling. The availability of cDNA clones for the alpha and beta subunits make it possible to study these structure-function relationships. While the invention has been described in conjunction with specific embodiments thereof, it is to be understood that other modifications are possible, and this patent application is intended to encompass all variations, applications or adaptations of the invention which generally follow the principles of the invention, including the deviations from the present invention Laid-open publication belonging to known or customary practice in the field to which the invention relates and how they can be applied to the essential features which the scope of the appended claims includes.

Claims (16)

A process for preparing an LFA-1 alpha subunit or its functional derivative, substantially free of natural contaminants, characterized in that a suitable host organism is an expression vector encoding, as a coding sequence, replicon and control sequences for expression in prokaryotes and Eukaryotes for the desired polypeptide at a suitable site, or the LFA-1. Alpha subunit or its functional derivative is chemically synthesized, and the desired polypeptide from the resulting transformants or the reaction mixture is isolated. 2. The method according to claim 1, characterized in that the LFA-1. Alpha subunit is also able to bind to a molecule present on the surface of a cell. 3. A method according to claim 2, characterized in that the molecule present on the surface of the cell is ICAM-1 or another natural ligand of LFA-1. 4. The method of claim 1, characterized in that the LFA-1 .Alpha subunit thus prepared can associate with an LFA-1 beta subunit to form an LFA-1 heterodimer. 5. The method according to claim 4, characterized in that the LFA-1 heterodimer is capable of binding to ICAM-1 or another natural ligand of LFA-1. 6. The method according to claim 5, characterized in that the coding sequence for the desired polypeptide contains at least one sequence which is selected from the following group: a. P-P-R-A-G-R-H; b. I-I-T-D-G-E-A; D-W-A-G-G-F-L; d. S-Q-V-Q-T-I-H; e. R-H-G-G-L-S-P; f. M-S-C-T-D-F-S; G. R-L-L-S-R-A-L; H. G-V-D-V-Q-D-G-E-I-E; i. D-I-N-G-D-G-L-V-D-V: j. V-K-D-L-E-G-D-G-L-A; k. T-Y-L-S-G-L; I. Y-I-I-G-I-G-K; m. I-E-G-T-Q-V-L-S-Q and n. P-S-I-H-N-I-P. 7. The method of claim 2 for the preparation of a functional derivative of an LFA-1 alpha subunit, characterized in that the coding sequence encodes a fragment of the LFA-1 alpha subunit. 8. The method according to claim 2 for the preparation of a functional derivative of an LFA-1 alpha subunit, characterized in that the coding sequence encodes a variant of the LFA-1 alpha subunit. 9. The method according to claim 2 for the preparation of a functional derivative of an LFA-1 alpha subunit, characterized in that the coding sequence encodes an analog of the LFA-1 alpha subunit. 10. The method according to claim 1, characterized in that the thus prepared LFA-1-alpha subunit or the functional derivative thereof is converted into a chemical functional derivative. A method of producing a DNA capable of expressing either the LFA-1 alpha subunit or one of its functional derivatives, characterized in that a suitable vector is digested with one or more suitable restriction endonucleases and the desired DNA is isolated becomes. 12. The method according to claim 11, characterized in that the DNA codes for an LFA-1 -Alphi: - subunit or its functional derivative containing at least one peptide sequence, which is selected from the following group .: a. P-P-R-A-G-R-H; b. M-T-D-G-E-A; c. D-W-A-G-G-F-L; d. S-Q-V-Q-T-I-H; e. R-H-G-G-L-S-P; f. M-S-C-T-D-F-S; G. R-L-L-S-R-A-L; H. G-V-D-V-Q-D-G-E-I-E; i. D-I-N-G-D-G-L-V-D-V; j, V-K-D-L-E-G-D-G-L-A; k. T-Y-L-S-G-L; I. Y-I-I-G-I-G-K; m. I-E-G-T-Q-V-L-S-Q and n. P-S-I-H-N-I-P. 13. A process for the preparation of a pharmaceutical composition for the treatment of inflammation resulting from a reaction of the specific defense system in mammals or metastases of a hematopoietic tumor cell, which cell requires a LFA-1 functional member to migrate, or growth of an LFA-1 alpha subunit expressing tumor cell, characterized in that an effective amount of a polypeptide according to claims 1 to 10 is incorporated into one or more fillers. 14. Use of a polypeptide according to claim 1 to 10, characterized in that it is used for the preparation of a pharmaceutical composition. A kit for determining the presence and location of inflammation resulting from a response of the mammalian specific defense system or the presence and location of a tumor cell expressing ICAM-1 or another natural ligand of LFA-1, characterized that it contains a detectably labeled LFA-1 alpha subunit or its derivative capable of identifying a cell expressing ICAM-1 or another natural ligand of LFA-1. A kit for determining the presence and location of inflammation resulting from a reaction of the specific defense system in a mammal, characterized in that it contains a nucleic acid molecule capable of binding to a molecule selected from the group which consists of an mRNA sequence of a gene for the LFA-1 alpha subunit, which binding is capable of identifying a cell that expresses ICAM-1 or another natural ligand of LFA-1.