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. This invention was made in part with government support. The government has certain rights in this invention.
. The present invention relates to the intercellular adhesion molecule, ICAM-1, and its ability to bind to the leukocyte adhesion receptor Mac-1. The invention further pertains to the use of the ICAM-1 - Mac-1 binding ability to treat inflammation.
. The immune system is responsible for protecting an animal from foreign invaders, such as bacteria, viruses, etc. An excellent review of the defense system is provided by Eisen, H. W. (In: Microbiology, 3rd Ed., Harper & Row, Philadelphia, PA (1980),
pp. 290-295 and 381-418). The ability of the immune system to protect an animal against foreign invaders depends, in large measure, on the presence and function of blood cells known an leukocytes.
. A primary event in the immune system's response to infectious agents is the recruitment of circulating neutrophils to the inflammatory site. Adhesion to the endothelium is the prerequisite physical step for extravasation to the peripheral site of
injury. Neutrophil localization has been examined on a molecular level to define both the sequence of events that promote neutrophil exit from the bloodstream and the cognate proteins on the surface of neutrophils and the endothelial cells that
coordinate this interaction. require the adhesion of immune system cells to cellular and extracellular substrates. For example, leukocytes must be able to attach to endothelial cells so that they can migrate from the circulation to sites of ongoing
inflammation. Furthermore, they must attach to antigen-presenting cells so that a normal immune response can occur. They must also be able to attach to appropriate target cells so that the lysis of virally-infected (or tumor) cells can occur.
Furthermore, leukocytes must be able to attach to various activated proteins (such as iC3b--the activated form of the third component of complement) so that they may efficiently phagocytose and clear microbial and cellular debris. Thus, leukocyte
adhesion is a requisite of a normally functioning host defense system The inhibition of this defense system is desirable in cases such as transplantation, because the host "sees" the transplanted tissue as foreign and initiates an immune response to that
tissue. Leukocyte adhesion is, therefore, also involved in the rejection of transplanted tissue and organs. Thus, an understanding of leukocyte adhesion may enable one to either augment an animal's ability to fight infection or suppress an animal's
capacity to reject transplanted tissue.
. Leukocyte surface molecules involved in mediating leukocyte adhesion were identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T-cells (Davignon, D., et al., Proc. Natl Acad. Sci. USA 78:4535-4539 (1981)) and
mouse spleen cells (Springer, T., et al., Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment-related functions described above (Springer, T., et al., Fed. Proc. 44:2660-2663 (1985)). The
molecules which were recognized by these antibodies comprise a set of leukocyte adhesion receptors known as the "Lymphocyte Function-Associated Antigen-1 family" (or the "LFA-1 family") of adhesion receptor molecules. related cell surface glycoproteins:
"LFA-1" (CD11a/CD18), "Mac-1" (CD11b/CD18), and "p150,95" (CD11c/CD18). These glycoproteins are members of a family of proteins, the leukocyte integrins, that are critical for adhesive functions in the immune system Springer et al. (Ann. Rev. Immunol,
5:223-252, (1987)).
. Whereas LFA-1 is found on the surfaces of most leukocytes (Springer, T. A., et al., Immunol. Rev. 68:111-135 (1982)), Mac-1 and p150,95 are found primarily on macrophages, granulocytes and other large granular lymphocytes (Springer, T. A., et al.,
Immunol. Rev. 68:111-135 (1982); Keizer, G., et al., Eur. J. Immunol. 15:1142-1147 (1985)).
. The LFA-1 family of adhesion receptor proteins are heterodimers which possess a common .beta. chain that is non-covalently associated with unique .alpha. chains. The alpha-subunits of the family have been found to differ from one another and are
designated CD11a, CD11b, and CD11c, respectively. The glycosylated alpha-subunits have approximate molecular weights of 175, 160, and 150 kd, respectively. In contrast, the beta-subunit of the LFA-1 family of adhesion receptors (designated "CD18") has
been found to be identical, and to have a molecular weight of 95 kd (Sanchez-Madrid, F., et al., J. Exper. Med. 158:1785-1803 (1983); Keizer, G. D., 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. 158:586-602 (1983)).
. Although the alpha-subunits of the glycoproteins do not exhibit the extensive homology shared by the beta-subunits, close analysis has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and
beta-subunits of the adhesion molecule glycoprotein family are provided by Sanchez-Madrid, F., et al. (J. Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983); Miller, L. J., et al., J. Immunol. 138:2381-2383 (1987)). studies which showed
the ability of monoclonal antibodies (which were capable of binding to either the specific alpha-subunits, or the common beta-subunit) to inhibit adhesion-dependent leukocyte functions (Sanchez-Madrid, F., et al., Proc. Natl. Acad. Sci. USA 79:7489-7493
(1982); Beller, D. I., et al., J. Exper. Med. 156:1000-1009 (1982)).
. The importance of the leukocyte integrins was confirmed by the discovery of a clinical syndrome, "Leukocyte Adhesion Deficiency" syndrome ("LAD"), that is characterized by a congenital deficiency or absence of the common .beta. (CD18) chain and
presents with diminished pus formation, abnormal wound healing, and grave susceptibility to pyrogenic infections (Anderson, D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al., J. Inf. Dis. 152:668-689 (1985); Anderson, D. C., et al..
Ann. Rev. Med. 38:175-194 (1987); Amaout, M. A., et al., J. Clin. Invest. 74:1291-1300 (1984)) as well as abnormalities of adhesion-dependent leukocyte functions in vitro (Anderson, D. C., et al., Ann. Rev. Med. 38:175-194 (1987); Todd, R. F., et al.,
Hem. Onc. Clinics N. A. 2:13-31 (1988)).
. Leukocytes from LAD patients display in vitro defects which were similar to those observed when leukocytes of normal individuals were antagonized by antibody specific for members of the LFA-1 family. LAD patients were found to be unable to mount a
normal immune response. This failure was found to be due to an inability of the leukocytes of LAD patients to adhere to 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 show that inflammatory reactions are mitigated when leukocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules on their cell surface.
. The three leukocyte adhesion proteins Mac-1, p150,95, and LFA-1 differ in function and in expression on leukocyte subpopulations. Mac-1 and p150,95 are expressed on neutrophils, and monocytes (Springer, T. A., et al., In: Biochemistry of Macrophages
(CIBA Symposium 118); Pitman, London, pp. 102-126 (1986)). During differentiation of blood monocytes into tissue macrophages, expression of p150,95 is greatly increased and Mac-1 expression is decreased (Schwarting, R., et al., Blood 65:974-983 (1985);
Hogg, N., et al., Eur. J. Immunol 16:240-248 (1986)). p150,95 is also expressed on certain types of activated T and B lymphocytes, but is not expressed on these cells in the blood (Kaligaris-Cappio, F., et al., Blood 66:1035-1042 (1985); Miller, L. J.,
et al., J. Immunol. 137:2891-2900 (1986); Keizer, G. D., et al., J. Immunol. 138:3130-3136 (1987)).
. LFA-1 is present on all leukocytes except 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, T. A., et al., Ann. Rev. Immunol 5:223-252 (1987)). Adhesion to the target cell is a step which is blocked by antibodies against LFA-1. Functional studies have suggested that LFA-1 interacts with several ligands, one of which is ICAM-1
(Rothlein, R., et al., J. Immunol. 137:1270-1274 (1986)).
. Mac-1 and p150,95 are expressed in an intracellular, vesicular compartment in circulating neutrophils and monocytes which is mobilized to the cell surface by inflammatory mediators (Todd, R. F., et al., J. Clin. Invest. 74:1280-1290 (1984);
Springer, T. A., et al., In: Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp. 102-126 (1986); Lanier, L. L., et al., Eur. J. Immunol. 15:713-718 (1985); Yancey, K. B., et al.. J. Immunol. 135:465-470 (1985). This mobilization
correlates with increased adhesiveness (Anderson, D. C., et al., Ann. Rev. Med. 38:175-194 (1987)). Mac-1 .alpha.-subunit message was detected in blood monocytes and PMA-induced myeloid cell lines, but not in cells of the T or B lineages, correlating
with Mac-1 protein surface expression.
. Some cytotoxic T lymphocyte clones have been found to express similar quantities of p150,95 and LFA-1. Monoclonal antibodies to the LFA-1 and p150,95 alpha-subunits inhibit killing by such CTL clones to similar extents and are additive in their
inhibitory effects (Keizer, G. D., et al., J. Immunol. 138:3130-3136 (1987)). Furthermore, antibodies to p150,95 alpha-subunits have been shown to inhibit monocyte attachment to endothelium (Keizer, G. D., et al., Eur. J. Immunol. 17:1317-1322 (1987)).
. Monoclonal antibodies to Mac-1 or p150,95 inhibit neutrophil aggregation and adherence to endothelial cells, protein-coated surfaces, bacteria, protozoan parasites, and fungi (Harlan, J. M., et al., Blood 66:167-178 (1985); Springer, T. A., et al.,
In: Biochemistry of Macrophages (CIBA Symposium 118), pitman, London, pp. 102-126 (1986); Dana, N., et al., J. Immunol. 137:3259 (1986); Bullock, W. D., et al., J. Exper. Med. 165:195-210 (1987); Mosser, D. M., et al., J. Immunol. 135:2785-2789 (1985)).
. MAC-1 (CD11b/CD18) is a leukocyte adhesion glycoprotein that has been demonstrated to bind multiple ligands including iC3b (Beller, D. I. et al., J. Exper. Med. 156:1000-1009 (1982)), fibrinogen (Altieri, D. C. et al., J. Cell. Biol. 107:1893-1900
(1988); Wright, S. D. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 85:7734-7738 (1988)),and Factor X (Altieri, D. C. et al.; J. Biol. Chem. 863:7007-7015 (1988)) in addition to its role in cell-cell and cell-substrate adhesive interactions. Detergent-soluble
Mac-1 and p150,95 have been shown to be able to bind to iC3b-Sepharose (Micklem, K. J., et al., Biochem. J. 231:233-236 (1985)).
. The .alpha.-subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains 3 putative divalent cation-binding sequences and 19
potential N-glycosylation sites. The amino acid sequence of Mac-1 .alpha. shows that it is a member of the integrin superfamily; Mac-1 .alpha. shows 63% identity to the .alpha.-subunit of the leukocyte adhesion glycoprotein p150,95 and 25% to the
.alpha.-subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor and the vitronectin receptor The Mac-1 .alpha.-subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree
of identity both to the p150,95 .alpha.-subunit (87% identity at the amino acid level) and to the rest of the integrin .alpha.-subunits (38%). The .alpha.-subunit of Mac-1, like the p150,95 .alpha.-subunit, contains a domain of 187 amino acids in the
extracellular region which is absent in other integrins. This inserted or "I" domain is homologous to the A domains of van Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw
attention to this region of Mac-1 as a potential binding site for iC3b.
. The functional role of Mac-1 was first illustrated by the ability of anti-Mac-1 .alpha.-subunit monoclonal antibodies (MAb) to block the rosetting of iC3b-coated erythrocytes to macrophages and polymorphonuclear leukocytes (Beller, D. I. et al., J.
Exper. Med. 156:1000-1009 (1982)), demonstrating that Mac-1 is indistinguishable from the complement receptor type three (CR3). Subsequently, the involvement of Mac-1 in inflammatory processes was evidenced by the inhibition of neutrophil aggregation and
adhesion to endothelial cells by anti-Mac-1 .alpha.-subunit and anti-b-subunit-specific MAb (Anderson, D. C. et al., J. Immunol. 137:15-27 (1986); Dana, N. et al., J. Immunol. 137:3259-3263 (1986); Vedder, N. B. et al., J. Clin. Invest. 81:672-682
(1988)). Recent epitope mapping studies have suggested that the sites involved in iC3b-binding are distinct from those involved in neutrophil aggregation and adherence to protein-coated plastic (Anderson, D. C. et al., J. Immunol. 137:15-27 (1986); Dana,
N. et al., J. Immunol. 137:3259-3263 (1986), Rosen, H. et al.. J. Exper. Med. 166:1685-1701 (1987)). Therefore, Mac-1 appears to be a multivalent receptor with at least two independent adhesion-related functions.
. The expression of functional activity of Mac-1 is regulated during leukocyte differentiation and activation. Differentiation and maturation of myelomonocytic cell lines results in increased Mac-1 expression (Miller, L. J. et al., J. Immunol.
137:2891-2900 (1986)), while blood monoctye differentiation into tissue macrophages is accompanied by a considerable decrease in the amount of Mac-1 on all cell surface (Hogg, N. et al., Eur. J. Immunol. 16:240-248 (1986)). The expression of Mac-1 on the
surface of circulating neutrophils and monocytes is upregulated by inflammatory stimuli; Mac-1 is stored in an intracellular vesicular compartment which is rapidly mobilized to the cell surface by chemoattractants (Todd, R. F. et al., J. Clin. Invest.
74:1280-1290 (1984)); Miller, L. J. et al., J. Clin. Invest. 80:535-544 (1987)). Although the augmented expression of Mac-1 can lead to increased adhesiveness, qualitative changes after cell activation may also be important in regulation ligand binding
(Detmers, P. A. et al., J. Cell Biol. 105:1137-1145 (1987)). Both the qualitative and quantitative changes may be important in regulation of leukocyte binding to post-capillary endothelium at inflammatory sites.
. The N-terminal sequence of the murine and human Mac-1 .alpha.-subunits (Miller, L. J. et al., J. Immunol. 138:2381-2383 (1987); Springer, T. A. et al., Nature 314:540-542 (1985)) and a murine genomic clone encoding a short N-terminal exon (Sastre,
L. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:5644-5648 (1986)) have been reported.
. Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells) and proteins (such as iC3b). This adherence has been found to require
contacts which involve specific receptor molecules present on the leukocyte surface of the leukocytes. These cell surface receptor molecules have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor
molecules exhibit chronic and recurring infections, as well as other clinical symptoms.
. Since leukocyte adhesion is involved in the process through which foreign tissue is identified and rejected, an understanding of this process is of significant value in the fields of organ transplantation, tissue grafts, allergy and oncology.
. The present invention relates to the cellular adhesion molecule, ICAM-1, and to the leukocyte cell surface adhesion receptor molecule Mac-1, and in particular, to the identification of the Mac-1 binding site on ICAM-1. The knowledge of the sequence
and structure of this binding site facilitates the use of Mac-1 and ICAM-1 functional derivatives in the treatment of inflammation.
. Previous studies with domain deletion and amino acid substitution mutants of ICAM-1 have localized the LFA-1 and human rhinovirus binding sites to the first NH.sub.2 -terminal Ig-like domain (Staunton, D. E., et al., Cell 61:243-254 (1990)). The
leukocyte integrin Mac-1, which is closely related to LFA-1, has also been shown to bind ICAM-1 (Smith, C. W., et al., J. Clin. Invest. 83:2008-2017 (1989).
. The present invention derives, in part, from a localization of the Mac-1 binding site on ICAM-1. Unexpectedly, a distinct binding region was found in the third NH.sub.2 -terminal Ig-like domain, and that two glycosylation sites in this domain
dramatically altered the ability of ICAM-1 to adhere to Mac-1.
. In detail, the invention pertains to an ICAM-1 functional derivative, especially a soluble functional derivative, which is substantially incapable of binding to Mac-1, but is substantially capable of binding to LFA-1.
. The invention specifically includes those of the above-described ICAM-1 functional derivatives which comprises Domains 1, 2, 3 and 4 of ICAM-1, and especially such derivatives which contain a mutation in Domain 3 of ICAM-1. Particularly preferred
mutations in Domain 3 are mutations in ICAM-1 residues 229-231, or 254-256 of ICAM-1.
. The present invention also provides an ICAM-1 functional derivative, especially a soluble derivative, which is substantially incapable of binding to LFA-1, but is substantially capable of binding to Mac-1. Among such derivatives are those which lack
Domain 1, of ICAM-1, and those which contain Domains 1, 2, 3, 4 and 5 of ICAM-1, but contain a mutation in Domains 1 or 2 of ICAM-1. Particularly preferred mutations are those in ICAM-1 residue(s) E34, Q58ED, or Q73 of Domain 1 of ICAM-1. Among preferred
ICAM-1 functional derivatives which lack Domain 1 are those comprising Domains 3 and 4 of ICAM-1.
. The invention also provides an ICAM-1 functional derivative which is substantially incapable of binding to Mac-1 and LFA-1, but is substantially capable of binding a human rhinovirus.
. The invention also pertains to an ICAM-1 functional derivative lacking a glycosylation site, wherein the derivative is capable of enhanced binding ability to Mac-1. Most preferred for this purpose is the loss of a glycosylation site at ICAM-1
residue N269, N240 or N358 of ICAM-1.
. The invention also provides an anti-inflammatory agent characterized in being capable of treating inflammation caused by a reaction of the non-specific defense system, but being substantially incapable of suppressing inflammation caused by a
reaction of the specific defense system.
. In particular, the invention provides an anti-inflammatory agent which is an ICAM-1 functional derivative, especially a soluble derivative, that is substantially incapable of binding to LFA-1, but is substantially capable of binding to Mac-1.
. The invention also provides an anti-inflammatory agent characterized in being capable of treating inflammation caused by a reaction of the specific defense system, but being substantially incapable of suppressing inflammation caused by a reaction of
the non-specific defense system.
. In particular, the invention provides an anti-inflammatory agent which is an ICAM-1 functional derivative, especially a soluble derivative, that is substantially incapable of binding to Mac-1, but is substantially capable of binding to
LFA-1.. |