Human CR3.alpha./.beta. heterodimers

Abstract

The invention features human CR3.alpha. recombinant or synthetic peptide capable of inhibiting a CR3-mediated immune response, a purified DNA encoding a human CR3.alpha. peptide, and a method of controlling any phagocyte-mediated tissue damage such as that associated with reduced perfusion of heart tissue during acute cardiac insufficiency. As used herein, a human CR3.alpha. recombinant peptide is a chain of amino acids derived from recombinant CR3.alpha.-encoding cDNA, or the corresponding synthetic DNA.

Description

BACKGROUND OF THE INVENTION

This invention relates to controlling the cellular immune/inflammatory responses, particularly phagocyte-mediated tissue injury and inflammation.

Circulating phagocytic white blood cells are an important component of the cellular acute inflammatory response. It is believed that a number of important biological functions such as chemotaxis, immune adherence (homotypic cell adhesion or aggregation), adhesion to endothelium, phagocytosis, antibody-dependent cellular cytotoxicity, and histaminase and lysosomal enzyme release are mediated by a leukocyte surface glycoprotein receptor, known as complement receptor type 3 (CR3), also known as Mol, CD11b/CD18, Mac-1 or MAM. See, Arnaout et al., J. Clin. Invest. 72:171-179 (1983), and references cited therein; Dana et al., J. Immunol. 137:3259-3263 (1986); Wallis et al., J. Immunol. 135:2323-2330 (1985); Arnaout et al., New Eng. J. Med. 312:457-462 (1985); Dana et al., J. Clin. Invest. 73:153-159 (1984); and Beatty et al., J. Immunol. 131:2913-2919 (1983).

CR3 consists of two noncovalently associated subunits. Kishimoto et al., Cell 48:681-690 (1987); Law et al., Eur. Mol. Biol. Organ. J. 6:915-919 (1987). The alpha subunit has an apparent molecular mass of 155-165 kD and associates in a divalent cation-independent manner with a beta subunit of 95 kD. Todd et al., Hybridoma 1:329-337 (1982). The beta subunit is common to two other leukocyte surface glycoproteins, LFA-1 and p150,95. In addition to sharing the property of binding to the same beta subunit, CR3, LFA-1, and p150,95 leukocyte adhesion molecules require divalent cations to mediate their adhesion functions, and they have homologous NH.sub.2 -termini. Pierce et al., Biochem. Biophys. Acta. 874:368-371 (1986); Miller et al., J. Immunol. 138:2381-2383 (1987).

Monoclonal antibodies have been used to identify at least two distinct functional domains of CR3, one mediating adhesion and the other mediating binding to the complement C3 fragment (iC3b). See, Dana et al., J. Immunol. 137:3259-3263 (1986).

Cosgrove et al., Proc. Nat'l. Acad. Sci. USA 83:752-756 (1986) report a human genomic clone which produces "a molecule(s)" reactive with monoclonal antibodies to CR3.alpha. (Mac-1 or OKM1).

Sastre et al., Proc. Nat'l. Acad. Sci. USA 83:5644-5648 (1986) report a mouse genomic clone coding for the .alpha. subunit of mouse complement receptor type 3.

It is believed that CR3 and p150,95 mediate enhanced adhesiveness of activated phagocytes through increased expression of these proteins on the surface of activated cells. For example, in granulocytes, these proteins are translocated from intracellular storage pools present in secondary and tertiary granules. Arnaout et al., J. Clin. Invest. 74:1291-1300 (1984); Arnaout et al., New Eng. J. Med. 312:457-462 (1985); Todd et al., J. Clin. Invest. 74:1280-1291 (1984).

Inherited deficiency of CR3 impairs leukocyte adhesion-dependent inflammmatory functions and predisposes to life-threatening bacterial infections. Dana et al., (1984), cited above.

Simpson et al., J. Clin. Invest. 81:624 (1988) disclose that a monoclonal antibody directed to an adhesion-promoting domain of CR3.alpha. reduces the extent of cardiac damage in dogs associated with myocardial infarction, presumably by limiting reperfusion injury.

SUMMARY OF THE INVENTION

The invention features human CR3.alpha. recombinant peptide. As used herein, human CR3.alpha. recombinant peptide is a chain of amino acids derived by expression of recombinant CR3.alpha.-encoding cDNA, or by expression of the corresponding synthetic DNA. For convenience, the term peptide is used to include all polypeptides regardless of length, including short polypeptides as well as proteins.

The invention also features human CR3.alpha. synthetic peptides that include at least one functional CR3 domain capable of inhibiting a CR3-mediated immune response. As used herein, "synthetic peptide" means a peptide derived by expression of recombinant DNA or by chemical synthesis. "CR3-mediated immune response" includes those functions mentioned above: chemotaxis, immune adherence (homotypic cell adhesion or aggregation), adhesion to endothelium, phagocytosis, antibody-dependent cellular cytotoxicity, and histaminase and lysosomal enzyme release. Inhibition of these immune functions can be determined by one or more of the following inhibition assays as described in greater detail below: cell-cell aggregation, phagocytosis, adhesion to endothelium, and chemotaxis.

Preferably, the peptide's functional domain has an amino acid sequence between 7 and 500 amino acid residues; most preferably, the domain is an amino acid sequence derived from the natural CR3.alpha. amino acid region that is homologous to a binding region of the yon Willebrand factor encompassing amino acids 134 through 340; or a peptide having one of the amino acid sequences: DIAFLIDGS; FRRMKEFVS; FKILVVITDGE; VIRYVIGVGDA; YYEQTRGGQVSVCPLPRGRARWQCDAV (fibronectin-like collagen binding domain; IL-2-receptor-like region); or KSTRDRLR. The peptide may also be derived from one or more of the metal binding domains of the CR3.alpha. peptide and preferably may be derived from the domains encompassing amino acids 358-412, 426-483, 487-553, or 554-614; most preferably, the peptide has one of the amino acid sequences: DVDSNGSTD, DVNGDKLTD, DLTMDGLVD, or DSDMNDAYL.

In another aspect, the invention features purified DNA encoding human CR3.alpha. or a synthetic CR3.alpha. peptide as defined above. The purified DNA may be cDNA encoding all of the CR3.alpha. peptide or a restriction enzyme fragment encompassing part of the CR3.alpha.-coding region, or synthetic DNA.

The invention can be used to control the cellular immune response mediated by CR3, where that response is not desired. The invention is particularly advantageous in that it avoids introduction of proteins of non-human species, such as non-human antibodies, which (particularly after the first administration) can raise undesired immune responses. It specifically provides a method of controlling damage associated with reduced perfusion of heart tissue (e.g. resulting from myocardial infarction) consisting of administering a therapeutic amount of the above described peptide in a suitable, pharmaceutically acceptable vehicle. It also can be used to treat dialysis leukopenia, a disease state characterized by phagocyte-dependent tissue injury.

In one particular embodiment, the peptide is administered as a heterodimer, with the human CR3.beta. peptide, preferably a truncated (soluble) CR3.beta. peptide that includes the extracellular position of the CR3.beta. peptide and lacks the transmembrane portion. Most preferably, the CR3.beta. portion of the heterodimer is a recombinant protein produced by expressing DNA encoding the truncated CR3.beta. peptide.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawing will first briefly be described.

Drawing

FIG. 1 is the cDNA sequence and deduced amino acid sequence of the open reading frame of human CR3.alpha. from Arnout et al., J. Cell. Biol. 106:2153-2158 (1988).

CR3.alpha. Peptide

The complete human CR3.alpha. peptide can be expressed from recombinant DNA encoding the entire .alpha. subunit. CR3.alpha. peptide fragments according to the invention can be expressed from a cloned restriction enzyme fragment of recombinant DNA or from synthesized DNA. The complete CR3.alpha. peptide can be obtained in the following way.

Isolation of a Human CR3.alpha. cDNA Clone

A 378 base pair (bp) cDNA clone encoding guinea pig CR3 was used as a probe to isolate three additional cDNA clones from a human monocyte/lymphocyte cDNA library as described in Arnaout et al., Proc. Nat. Aca. Sci. 85:2776 (1988); together these three clones contain the 3,048-base nucleotide sequence encoding the CR3.alpha. gene shown in FIG. 1.

In order to express CR3.alpha., a mammalian expression vector can be constructed by assembling the above-described three cDNA clones. Appropriate restriction enzyme sites within the CR3.alpha. gene can be chosen to assemble the cDNA inserts so that they are in the same translation reading frame. A suitable basic expression vector can be used as a vehicle for the 3,048 bp complete cDNA fragment encoding the human CR3.alpha. peptide; the recombinant cDNA can be expressed by transfection into, e.g., COS-1 cells, according to conventional techniques, e.g., the techniques generally described by Aruffo et al., Proc. Nat'l. Acad. Sci. USA 84:8573-8577 (1987).

Isolation of CR3.alpha. Peptide from Mammalian Cells

The CR3.alpha. protein can be purified from the lysate of transfected COS-1 cells, using affinity chromatography and lentil-lectin Sepharose and anti-CR3.alpha. monoclonal antibody as described by Pierce et al. (1986) cited above and Arnaout et al., Meth. Enzymol. 150:602 (1987). Anti-CR3.alpha. monoclonal antibody can be made according to techniques well known in the art using synthetic peptides corresponding to functional domains of the CR3.alpha. molecule as the initial immunogen, or it can be purchased from Becton Dickinson, Ortho Pharmaceutical, or other sources.

If the desired CR3.alpha. peptide is shorter than the entire protein, DNA encoding the desired peptide can be expressed in the same mammalian expression vector described above using the selected DNA fragment and the appropriate restriction enzyme site, as outlined above. The selected DNA fragment may be isolated according to conventional techniques from one of the CR3.alpha. cDNA clones or may be synthesized by standard phosphoamidite methods, as described by Beaucage et al., Tetrahedron Letters 22:1859 (1981).

Characterization of the CR3a Polypeptide

The coding sequence of the complete CR3.alpha. protein is preceded by a single translation initiation methionine. The translation product of the single open reading frame begins with a 16-amino acid hydrophobic peptide representing a leader sequence, followed by the NH.sub.2 -terminal phenylalanine residue. The translation product also contained all eight tryptic peptides isolated from the purified antigen, the amino-terminal peptide, and an amino acid hydrophobic domain representing a potential transmembrane region, and a short 19-amino acid carboxy-terminal cytoplasmic domain (FIG. 1). The coding region of the 155-165 kD CR3.alpha. (1,136 amino acids) is eight amino acids shorter than the 130-150 kD alpha subunit of p150,95 leukocyte adhesion molecule (1,144 amino acids). The cytoplasmic region of CR3.alpha. contains one serine residue that could serve as a potential phosphorylation site. The cytoplasmic region is also relatively rich in acidic residues and in proline (FIG. 1). Since CR3 is involved in the process of phagocytosis and is also targeted to intracellular storage pools, these residues are candidates for mediating these functions. The long extracytoplasmic amino-terminal region contains four metal-binding domains (outlined by broken lines in FIG. 1) that are similar to Ca.sup.2+ -binding sites found in other integrins. Each metal binding site may be composed of two noncontiguous peptide segments and may be found in the four internal tandem repeats formed by amino acid residues 358-412, 426-483, 487-553, and 554-614. The extracytoplasmic region also contains a unique 187-amino acid sequence, beginning at residue 151, which is not present in the homologous alpha subunits of fibronectin, vitronectin, or platelet IIb/IIIa receptors. This sequence is present in the highly homologous alpha subunit of leukocyte p150,95 with 57% of the amino acids identical and 34% representing conserved substitutions. It is known that both Mol and p150,95 have a binding site for complement fragment C3 and this unique region may be involved in C3 binding. This region of CR3.alpha. also has significant homology (17.1% identity and 52.9% conserved sustitutions) to the collagen/heparin/platelet GpI binding region of the mature von Willebrande factor (residues 530-713).

The following peptides can be used to inhibit CR3 activity: a) peptide identical to the above-described p150,95-homologous region of CR3.alpha., or a domain thereof, e.g., DIAFLIDGS, FRRMKEFVS, FKILVVITDGE, or VIRYVIGVGDA; b) YYEQTRGGQVSVCPLPRGRARWQCDAV (fibronectin-like collagen binding domain, IL-2-receptor-like region); c) peptides identical to one or more of the four metal binding regions of CR3.alpha., e.g., having one of the following amino acid sequences: DVDSNGSTD, DVNGDKLTD, DLTMDGLVD, DSDMNDAYL; d) peptides substantially identical to the complete CR3.alpha.; or e) other CR3.alpha. domains, e.g. KSTRDRLR.

A CR3.alpha. peptide, such as one of those described above, can be tested in vitro for inhibition in one of the following five assays: iC3b binding; cell-cell aggregation, phagocytosis, chemotaxis, or adhesion to endothelium; or tested in vivo for controlling damage associated with reduced perfusion of heart tissue, as a result of myocardial infarction.

Inhibition of Granulocyte or Phagocyte Adhesion to iC3b-Coated Erythrocytes or Bacteria

The antimicrobial activity of the neutrophil depends to a significant degree on the ability of this cell to establish a firm attachment to its target. For this purpose, neutrophils possess a number of specific cell surface receptors that promote this interaction, such as a receptor which binds to complement C3 (iC3b), e.g. the CR3 receptor. Human neutrophilic polymorphonuclear granulocytes can be isolated from EDTA-anticoagulated blood on Ficoll-Hypaque gradients (Boyum, Scand. J. Clin. Invest. (Suppl.) 21:77 (1968)) modified as described by Dana et al., J. Clin. Invest. 75.:153 (1984)). Phagocytes can be prepared by incubating the mononuclear cell fraction (obtained from Ficoll-Hypaque centrifugation) on plastic petri dishes (Todd et al., J. Immunol. 126:1435 (1981)). Peptides of the invention can be tested for their ability to inhibit iC3b mediated binding of granulocytes to sheep erythrocytes as described in Dana et al., 1984, cited above and Arnaout et al., (1985) cited above.

Inhibition of Phagocytosis

Phagocytosis is an important biological function resulting in clearing of damaged tissue from the body, and in elimination of foreign particles (bacteria, fungi). An in vitro test for inhibition of phagocytosis is described in Arnaout et al., New Eng. J. Med. 306:693 (1982).

Inhibition of Phagocyte Adhesion to Endothelium

Monocytes must cross vascular endothelium during their egress from blood to extravascular tissues. Studies of leukocyte kinetics in animals indicate that acute inflammatory reactions may be marked by a massive increase in transendothelial monocyte/granulocyte traffic. In many chronic inflammatory lesions, perivascular monocytes accumulate in skin windows more slowly than neutrophils, but later become the predominant cell type. In addition monocytes leaving the circulation can rapidly acquire the morphoology of resident tissue macrphages--in some cases within a few hours of their departure from plasma. Thus, vascular endothelium may be considered an important substrate with which monocytes (and granulocytes) must interact during adherence, diapedesis, and differentiation. An in vitro assay for monocyte/granulocyte interaction with the vessel wall consists of binding radiolabeled monocyte/granulocyte preparations to cultured vascular endothelium, as described in Mentzer et al., J. Cell Physiol. 125:285 (1986).

Inhibition Of Chemotaxis

The ability of cells of the immune system to migrate is essential to the cellular immune response that results in tissue inflammation. Therefore, a peptide of the invention can be tested for its ability to inhibit chemotaxis, as described in Dana et al., (1986), cited above.

Cell-Cell Agregation

Granulocyte aggregation will be performed as detailed elsewhere. Arnaout et al., New Engl. J. Med. (1985) cited above. Aggregation will be induced by zymosan-activated autologous serum or with chemotactic peptides, e.g. FMLP. Aggregation will be recorded as incremental change in light transmission [.DELTA.T] using a platelet aggregometer. The reading can be confirmed by phase microscopy.

In Vivo Model for Testing Peptide

Damage to the heart tissue during myocardial infarction can be minimized by administering to an animal an inhibitor of the CR3-mediated immune response. A peptide of the invention may be tested for in vivo effectiveness using animals, e.g., dogs, which have been induced to undergo myocardial infarction. See, e.g. Simpson et al. cited above.

Use

The peptide can be administered intravenously in saline solution generally on the order of mg quantities per 10 kilograms of body weight. The peptide can be administered in combination with other drugs, for example, in combination with, or within six hours to three days after a clot dissolving agent, e.g., Tissue Plasminogen Activator (TPA), Activase, or Streptokinase.

Heterodimer

It may be advantageous to administer the heterodimer formed by the CR3.alpha. and CR3.beta. proteins. Expression of the CR3.alpha. chain is described elsewhere in this application. Expression of the CR3.beta. chain has been reported by others. See, e.g. Law et al. EMBO J. 6:915-919 (1987); Kishimoto et al. Cell 48:681-690 (1987); Tankum et al. Cell 46:271-282 (1986). The strategies described above or in those reports can be used to obtain CR3.beta. to make such a heterodimer.

Preferably, a secreted form of CR3.alpha./CR3.beta. complex can be produced by co-transfecting COS cells using the cloned CR3.alpha. described above and the cloned CR3.beta.. A secreted form of the complex can be produced by known techniques by generating stop codons 5' to the DNA sequences encoding the transmembrane regions of the two subunits. Culture supernatants from COS cells transfected with the combined cDNAs will be used, as described elsewhere, preferably after purification.

Other emobodiments are within the following claims.

Claims

1. A recombinant human CR3 heterodimer comprising

a) a soluble human CR3.alpha. peptide comprising the entire extracellular domain of human CR3.alpha., and being capable of inhibiting a CR3-mediated immune response, said CR3.alpha. peptide lacking any of the transmembrane domain of CR3.alpha. and said CR3.alpha. peptide lacking any of the cytoplasmic domain of CR3.alpha., and

b) a soluble human CR3.beta. peptide, said human CR3.beta. peptide comprising the extracellular domain of CR3.beta., and lacking any of the transmembrane domain of CR3.beta. and said CR3.beta. peptide lacking any of the cytoplasmic domain of CR3.beta.,

said heterodimer being capable of inhibiting a CR3-mediated immune response.

2. A composition comprising the human CR3 heterodimer of claim 1 and a physiologically acceptable carrier.